Cause Of Foodborne Disease Outbreaks Research Articles

Tolerance Increase in Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus Aureus USA300 Exposed to Low-Power Continuous Ultraviolet Radiation from Narrow-Wavelength Sources

Escherichia coli O157:H7 is a major cause of foodborne disease outbreaks throughout the world, while methicillin-resistant Staphylococcus aureus (MRSA) is responsible for many difficult-to-treat infections in humans. Ultraviolet (UV) irradiation is commonly used for disinfection in food processing, medical facilities, and water treatment to prevent the transmission of these pathogen. With increased use of UV disinfection technologies over the last few years because of COVID-19 and concerns about other communicable disease, it has become a concern that microbial species could develop tolerance to UV irradiation, especially when it is applied continuously. To elucidate the effect of continuous UV exposure at different wavelengths and power levels on the tolerance development of bacteria, Escherichia coli O157:H7 and MRSA)USA300 growths were investigated by continuously exposing inoculated agar plates to six different commercially available UV sources at wavelengths of 222 nm, 254 nm, 275 nm, and 405 nm. The agar plates in these experiments were partially covered by a thin acrylic sheet, which provided either complete protection from the UV to the cells directly under the sheet, no protection if significantly away from the sheet, or partial protection near the edges of the sheet due to shading or small amounts of UV reflection under the sheet at the edges. In these experiments, tolerant cells of E. coli and S. aureus were found from the 222 nm, the 405 nm, and one of the 254 nm sources. Upon examination of the power of each UV source, it was shown that the 275 nm and 254 nm sources that resulted in no tolerant cells had surface power densities [at 25 cm (10 in.)] that were more than 10–200 times greater than those that had tolerant cells. These results suggests that bacterial cells have a higher chance to develop UV tolerance under lower power UV sources (under the experimental conditions in our laboratory). Genome investigation of the tolerant colonies revealed that there are no significant differences between the cells that developed tolerance and the original organism, hinting at the need to explore the role of epigenetics mechanisms in the development of UV tolerance in these bacteria.

A systematic literature review of milk consumption and associated bacterial zoonoses in East Africa.

Consumption of unsafe animal-source foods is the major cause of foodborne disease outbreaks in low-income countries. Despite current knowledge of the threat posed by raw milk consumption to human health, people in many countries in East Africa still consume unboiled milk. This literature review explored the association between milk consumption and the occurrence of five milk-borne bacterial zoonoses: brucellosis, salmonellosis, campylobacteriosis, Escherichia coli infections, and tuberculosis. A search for literature published up to 1 October 2021 was conducted through the Web of Science, PubMed, and Scopus databases, using Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. The selection process yielded 65 articles describing studies conducted in East Africa 2010-2021, which were carefully scrutinized. The most investigated pathogen was Brucella spp. (54.5%), followed by E. coli (18.2%), Salmonella spp. (12.1%), Mycobacterium spp. (6.1%), and E. coli O157: H7 (6.1%). The most common predisposing factors for potential milk-borne disease outbreaks were consumption of contaminated raw milk, inadequate cold storage along the milk value chain, poor milk handling practices, and lack of awareness of the health risks of consuming unpasteurized milk. Thus, a tailor-made training program is needed for all milk value chain actors to enhance the safety of milk sold in informal markets, and a One Health approach should be applied. Future studies should employ more advanced diagnostic techniques and countries in East Africa should invest in modern diagnostic tools and equipment, both in hospitals and in local rural settings where most cases occur.

Chicken Production and Human Clinical Escherichia coli Isolates Differ in Their Carriage of Antimicrobial Resistance and Virulence Factors.

Contamination of food animal products by Escherichia coli is a leading cause of foodborne disease outbreaks, hospitalizations, and deaths in humans. Chicken is the most consumed meat both in the United States and across the globe according to the U.S. Department of Agriculture. Although E. coli is a ubiquitous commensal bacterium of the guts of humans and animals, its ability to acquire antimicrobial resistance (AMR) genes and virulence factors (VFs) can lead to the emergence of pathogenic strains that are resistant to critically important antibiotics. Thus, it is important to identify the genetic factors that contribute to the virulence and AMR of E. coli. In this study, we performed in-depth genomic evaluation of AMR genes and VFs of E. coli genomes available through the National Antimicrobial Resistance Monitoring System GenomeTrackr database. Our objective was to determine the genetic relatedness of chicken production isolates and human clinical isolates. To achieve this aim, we first developed a massively parallel analytical pipeline (Reads2Resistome) to accurately characterize the resistome of each E. coli genome, including the AMR genes and VFs harbored. We used random forests and hierarchical clustering to show that AMR genes and VFs are sufficient to classify isolates into different pathogenic phylogroups and host origin. We found that the presence of key type III secretion system and AMR genes differentiated human clinical isolates from chicken production isolates. These results further improve our understanding of the interconnected role AMR genes and VFs play in shaping the evolution of pathogenic E. coli strains. IMPORTANCE Pathogenic Escherichia coli causes disease in both humans and food-producing animals. E. coli pathogenesis is dependent on a repertoire of virulence factors and antimicrobial resistance genes. Food-borne outbreaks are highly associated with the consumption of undercooked and contaminated food products. This association highlights the need to understand the genetic factors that make E. coli virulent and pathogenic in humans and poultry. This research shows that E. coli isolates originating from human clinical settings and chicken production harbor different antimicrobial resistance genes and virulence factors that can be used to classify them into phylogroups and host origins. In addition, to aid in the repeatability and reproducibility of the results presented in this study, we have made a public repository of the Reads2Resistome pipeline and have provided the accession numbers associated with the E. coli genomes analyzed.

Epidemiological and etiological characteristics of Vibrio parahaemolyticus strains causing foodborne disease outbreaks in Guangdong Province from 2017 to 2020

Objective: To study the epidemiological and pathogenic characteristics of Vibrio parahaemolyticus isolated from outbreaks cases in Guangdong Province, 2017-2020. Methods: Epidemiological characteristics of 87 outbreak events caused by Vibrio parahaemolyticus were analyzed. Strains were serotyped, and then analyzed by pulsed-field gel electrophoresis (PFGE). Results: The food-borne disease outbreak caused by Vibrio parahaemolyticus was found in 16 cities. 44.8% (39/87) and 37.9% (33/87) of the outbreaks occurred in hotels, restaurants and school canteens, respectively. Improper food processing and storage (40.2%, 35/87) and cross contamination caused by indiscriminate raw and cooked food (25.3%, 22/87) were the main causes of food-borne disease outbreaks of Vibrio parahaemolyticus. The main serotypes of patient derived strains were O3:K6 (87.5%) and O4:KUT (22.5%). The similarity value between O3:K6 type isolates was 65.5%-100.0%, and the PFGE pattern similarity value of O4:KUT type isolates was 66.5%-100.0%. Conclusion: Outbreaks caused by Vibrio parahaemolyticus are widely distributed in Guangdong province. It is necessary to strengthen the publicity and education on the correct handling of food in hotels, restaurants, schools, and unit canteens. O3:K6 and O4:KUT serotypes are the main serotypes of the outbreak. There is genetic diversity among the epidemic strains.

Antibiotic Resistance and Plasmid Profile Analysis of Salmonella Enteritidis Isolated in Siberia and the Far East of Russia between 1990 and 2017.

Salmonella is one of the major causes of foodborne disease outbreaks globally. Specifically, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the major causes of zoonotic Salmonella infection in humans worldwide. In this study, we present data on antimicrobial resistance (AMR) and plasmid profiles of S. Enteritidis strains isolated from patients, food, and the environment in Siberia and the Far East of Russia obtained during Salmonella monitoring between 1990 and 2017. A total of 345 S. Enteritidis isolates were tested by the disk diffusion method with a set of 15 antibiotics using EUCAST breakpoints v. 10 and by plasmid profile analysis using the alkaline lysis method. The results have shown a substantial decrease in susceptibility to aminoglycosides and quinolones during the study period. No significant differences were found in the susceptibility of strains between regions as well as in the its correlation with different plasmid types of the pathogen. Several S. Enteritidis strains were found to be resistant to ampicillin, kanamycin, tetracycline, chloramphenicol, and cephalosporins. All tested S. Enteritidis strains were susceptible only to imipenem. In this study, we observed a relatively low level of AMR in S. Enteritidis strains isolated in Siberia and the Far East of Russia. Nevertheless, it is important to continue the molecular genetic monitoring and AMR surveillance of S. Enteritidis to track further increases in AMR using conventional phenotypic susceptibility testing and by introducing whole-genome sequencing to identify AMR mechanisms.

Endogenous Metabolites Released by Sanitized Sprouting Alfalfa Seed Inhibit the Growth of Salmonella enterica.

Sprouts are the leading cause of foodborne disease outbreaks globally, mainly because the specialized conditions required to germinate seed sprouts for human consumption contribute to an environment that allows pathogenic bacteria to flourish. To reduce risk of illness, current food safety guidelines in the United States and Canada recommend hypochlorite treatment for seed sanitation. However, many growers and consumers have become wary of the impact of hypochlorite on human health and the environment and are actively seeking less caustic approaches. Here, we evaluated the effects of both the traditional hypochlorite treatment and a milder alternative on nontyphoidal Salmonella enterica colonization of germinating alfalfa seed. Moreover, we explored three biological factors as potential contributors for inhibition of S. enterica growth: colonization by indigenous bacteria, seed composition changes, and seed metabolite release. In this experimental setting, we found that a combinatorial treatment of heat, peroxide, and acetic acid was as effective as hypochlorite for inhibiting S. enterica growth. Notably, we pinpointed N-acetyl-spermidine as an endogenous metabolite exuded by treated seeds that strongly inhibits S. enterica growth. In doing so, we both elucidated one of the mechanisms of chemical sanitation and highlighted a potential seed-derived mode of antimicrobial treatment that may apply to modernized food safety protocols.IMPORTANCE Warm, humid, and nutrient-rich conditions that are used to produce sprouts encourage Salmonella enterica to proliferate. However, many disparate sanitation methods exist, and there is currently no single treatment that can guarantee pathogen-free seeds. Here, we compared the ability of traditional hypochlorite treatment against a combinatorial treatment of heat, peroxide, and vinegar (HPA) commonly used in organic farming practices to inhibit S. enterica colonization and growth during alfalfa germination and found HPA to be at least as effective. Furthermore, we explored seed-based changes following sanitization treatments using metabolomics and identified polyamines as strong inhibitors of Salmonella growth on germinating alfalfa. Our findings enable a better understanding of host-pathogen interactions in sprout microbial communities and promote in-depth, evidence-based research in seed sprout safety.

Mushroom Poisoning Outbreaks - China, 2010-2020.

Introduction: Mushroom poisoning was the leading cause of foodborne disease outbreaks and outbreak-associated deaths in China. Mushroom poisoning outbreak surveillance can provide insight into the epidemiological characteristics of mushroom poisonings and guide policymaking and health education to reduce illnesses and deaths. Methods: Foodborne Disease Outbreak Surveillance System was upgraded in 2011 to collect foodborne disease outbreaks in China. Mushroom poisoning outbreaks during 2010–2020 were selected to analyze geographical distribution, seasonal distribution, and setting of food preparation. Results: A total of 10,036 outbreaks, which resulted in 38,676 illnesses and 788 deaths, were reported in this period. Mushroom poisonings occurred all over the country, but with highest incidence in the southwest and central China. Overall, 84.6% outbreaks were associated with food prepared in households, followed by 8.7% in street stalls, and 2.5% in canteens. Mushroom poisoning outbreaks clearly exhibited seasonality, and the peak season was summer through autumn. Outbreaks occurring between May and October accounted for 94.1% of total outbreaks, 92.4% illnesses, and 97.2% deaths. Conclusions: Mushroom poisoning is a food safety issue of higher concern in China. Targeted health education is essential to reduce mushroom poisoning, especially in southwest China. Citizens are advised to not collect or eat wild mushrooms.

Associations between climatic parameters and the human salmonellosis in Yazd province, Iran

Salmonella is one of the most common causes of foodborne disease outbreaks in developing countries. Climatic factors such as temperature, rainfall, and relative humidity can directly increase the growth and spread of these pathogens. Therefore, the aim of this study was to investigate long-term temporal trends and seasonal patterns of Salmonella infections as well as evaluating the effects of demographic and climatic factors on the infection incidence in Yazd province, Iran during 2012–2015. The incidence of Salmonella infections was highest among patients with the age group of ≤5 years and peaked in summer, especially during June. Contrary to expectations, no significant associations were seen between the average monthly temperature, rainfall or humidity and incidence rate (IR) of salmonellosis. Interestingly, atmospheric dust hovering was significantly associated with an increased risk of salmonellosis. Transmission pathways of Salmonella spp. in communities should be considered as a complex ecological process that animal reservoirs, socio-economic factors, and lifestyle behaviors need to be addressed in future studies.

Molecular Epidemiology of GI.3 Norovirus Outbreaks from Acute Gastroenteritis Surveillance System in Taiwan, 2015–2019

Norovirus is the leading cause of food-borne disease outbreaks. We conducted this study to examine the incidence and molecular characteristics of norovirus genogroup I infections from acute gastroenteritis outbreaks in Taiwan. Between January 2015 and June 2019, 2121 acute gastroenteritis clusters were reported to Taiwan CDC, of which 351 (16.5%) clusters were positive for NoV GI, and GI.3 was the most prevalent (36.8%) during the study period. The GI.3 infections were significantly higher than non-GI.3 infections in the age groups of 0–5 and 6–18 years. The phylogenetic analysis of the MCC tree revealed that VP1 genes were divided into 3 groups: the GI.P3-GI.3 strains in Taiwan were genetically close to Japan and the GI.Pd-GI.3 strains were segregated into 2 other groups which were genetically closely related to China. In addition, 7 GI.Pd-GI.3 recombinants were identified circulating in Taiwan between 2018 and 2019, and the prevalence of GI.Pd-GI.3 should be monitored to assess whether this could become the new predominant strains in neighboring Asian countries or other parts of the world. Both GI.P3-GI.3 and GI.Pd-GI.3 strains cocirculate, the recombination among these two lineages occurs frequently, contributing to the genetic diversity and multiple occurrences of different norovirus lineages, and their rapid evolution makes future control more difficult. Continued surveillance and timely interventions are critical to understand the complexity of norovirus gene variation and to monitor the new emerging norovirus strains.

Probabilistic risk model of norovirus transmission during handling and preparation of fresh produce in school foodservice operations

Human noroviruses (NoV) are recognized worldwide as important pathogens and the primary cause of foodborne disease outbreaks from contaminated food in the U.S. They are often transmitted by infected food handlers manipulating foods during preparation, such as fresh fruits and vegetables. This paper provides a study to model the transfer of NoV between food handlers and vegetables during salad preparation in school food services based on direct observation data. Three transfer pathways were modeled by considering different initial contamination sources (environment, handlers and contaminated produce). The probability of infection by NoV was also estimated based on the NoV levels at consumption obtained from each simulated transfer pathway. A scenario analysis ranging a wide concentration from 102 to 107 NoV infective particles was performed to represent different levels of NoV in the initial contamination sources. In addition, a sensitivity analysis was applied to identify the most important model inputs and determine the safest handling practices to be implemented in school food service operations. The pathway describing transfer from contaminated surfaces or handlers to foods indicated that initial levels of ≤104 NoV particles/fomite resulted in <0.5% cases per serving of NoV infection. When initial levels were higher, % cases of NoV infection was estimated to be ca. 3%. This rise in % cases of infection was linked to higher doses (5% serving with ≥15 NoV particles/serving) and prevalence levels at consumption (>0.2). In the pathway modeling cross contamination from contaminated vegetables to non-contaminated vegetables, all scenarios could lead to infected individuals, although number of cases of infection were lower (<1.3%), despite concentration levels were higher. On the contrary, for this pathway, prevalence was 2-fold lower than that observed in the pathways describing transfer from contaminated surfaces and hands. Based on the sensitivity analysis, NoV transfers to fresh produce may be minimized by improving hand washing, and therefore effective training programs need to be carried out specifically addressing hand washing. Moreover, the produce's washing step showed to be an effective control measure, depending on the desinfectant efficacy, by reducing % cases of NoV infection from 6 to 1%. The model in this study might be used, in the future, to evaluate the impact on the risk associated with NoV transmission of specific and effective training programs, aimed at food chain operators.

Surveillance for Foodborne Disease Outbreaks - United States, 2009-2015.

Problem/ConditionKnown foodborne disease agents are estimated to cause approximately 9.4 million illnesses each year in the United States. Although only a small subset of illnesses are associated with recognized outbreaks, data from outbreak investigations provide insight into the foods and pathogens that cause illnesses and the settings and conditions in which they occur.Reporting Period2009–2015Description of SystemThe Foodborne Disease Outbreak Surveillance System (FDOSS) collects data on foodborne disease outbreaks, which are defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food. Since the early 1960s, foodborne outbreaks have been reported voluntarily to CDC by state, local, and territorial health departments using a standard form. Beginning in 2009, FDOSS reporting was made through the National Outbreak Reporting System, a web-based platform launched that year.ResultsDuring 2009–2015, FDOSS received reports of 5,760 outbreaks that resulted in 100,939 illnesses, 5,699 hospitalizations, and 145 deaths. All 50 states, the District of Columbia, Puerto Rico, and CDC reported outbreaks. Among 2,953 outbreaks with a single confirmed etiology, norovirus was the most common cause of outbreaks (1,130 outbreaks [38%]) and outbreak-associated illnesses (27,623 illnesses [41%]), followed by Salmonella with 896 outbreaks (30%) and 23,662 illnesses (35%). Outbreaks caused by Listeria, Salmonella, and Shiga toxin-producing Escherichia coli (STEC) were responsible for 82% of all hospitalizations and 82% of deaths reported. Among 1,281 outbreaks in which the food reported could be classified into a single food category, fish were the most commonly implicated category (222 outbreaks [17%]), followed by dairy (136 [11%]) and chicken (123 [10%]). The food categories responsible for the most outbreak-associated illnesses were chicken (3,114 illnesses [12%]), pork (2,670 [10%]), and seeded vegetables (2,572 [10%]). Multistate outbreaks comprised only 3% of all outbreaks reported but accounted for 11% of illnesses, 34% of hospitalizations, and 54% of deaths.InterpretationFoodborne disease outbreaks provide information about the pathogens and foods responsible for illness. Norovirus remains the leading cause of foodborne disease outbreaks, highlighting the continued need for food safety improvements targeting worker health and hygiene in food service settings. Outbreaks caused by Listeria, Salmonella, and STEC are important targets for public health intervention efforts, and improving the safety of chicken, pork, and seeded vegetables should be a priority.Public Health ActionThe causes of foodborne illness should continue to be tracked and analyzed to inform disease prevention policies and initiatives. Strengthening the capacity of state and local health departments to investigate and report outbreaks will assist with these efforts through identification of the foods, etiologies, and settings linked to these outbreaks.

Analysis of frozen strawberries involved in a large norovirus gastroenteritis outbreak using next generation sequencing and digital PCR

Virus-contaminated frozen berries have been frequently identified as cause of foodborne disease outbreaks. To provide new tools for virus detection and characterization in berries, next generation sequencing (NGS) and reverse transcription-digital PCR (RT-dPCR) techniques were tested here with strawberries previously involved in a large-scale norovirus (NoV) gastroenteritis outbreak in Germany. By NGS, about 29 million sequence reads were generated, which mainly showed identities to sequences from the plant matrix and from the bacterial flora. Most abundant virus sequences originated from plant-specific viruses, whereas sequences with high identity to human viruses were rare. Only two sequence reads showed homologies to human NoV. They were identical to GII.P16/GII.13 NoV sequences from patients and a strawberry sample independently analyzed during the outbreak. Quantification of the GII NoV RNA of the berries using RT-dPCR confirmed a low mean virus amount of 185 copies/25 g, which is similar to independently assessed RT-qPCR results (257 copies/25 g). The study shows that identification of human-pathogenic viruses in naturally contaminated frozen berries is possible using NGS technologies. However, the method needs to be further optimized in order to enable convenient and reproducible detection of a low amount of human-pathogenic virus sequences in a background of highly abundant nucleic acids of other sources.

Salmonella Colitis: An Important Differential Diagnosis of Inflammatory Bowel Disease

26 year old man presented to GI clinic with a complaint of severe bilateral lower abdominal pain associated with fever, night sweats and rectal bleeding for several days. Abdominal pain was sharp, 10/10 in intensity with no obvious relieving and aggravating factors. He had underwent colonoscopy one month previously due to intermittent rectal bleed for several weeks which revealed an anal fissure with no evidence of colitis. Conservative management with suppositories resulted in resolution of bleeding. He was admitted for additional workup. CT scan showed thickening of the left colon extending into recto-sigmoid area. IBD was high in the differential diagnosis. Flexible sigmoidoscopy was done which showed severe ulcerative colitis (UC) in descending and sigmoid colon as well as rectum. He was started on IV Solumedrol empirically. He did not show much improvement over the next 24 hours. On day 2 his stool culture was positive for salmonella species and he was started on Levofloxacin and steroids were stopped. His symptoms improved. Pathology showed acute colitis with no chronic architectural changes. He was discharged on day 4 in clinically stable condition with diagnosis of Salmonella colitis. Follow up in GI clinic showed remarkable improvement. Discussion: Approximately 200 to 300 cases of Salmonella are reported in the USA each year. S. enteritidis, a common cause of foodborne disease outbreaks, is most frequently associated with eggs or egg-containing products. Salmonella infection usually affects the Small Intestine, but it can also affect the colon resulting in diffuse colitis. The colitis may be difficult to differentiate from UC. Stool studies should always be ordered and infectious etiologies should be ruled out, even in patients with known IBD who present with worsening symptoms. Colonoscopy is not necessary for diagnosis of Salmonella Colitis, as diffuse colitis is seen in both UC and Salmonella Colitis. Pathology may assist with the diagnosis, however. Treatment has traditionally been a Fluoroquinolone, but severe cases may require the addition of Ceftriaxone due to increasing resistance patterns. Our case report discusses a young man with symptoms and sigmoidoscopy findings suggestive of UC. Stool studies showed the unexpected presence of salmonella. This case reinforces the importance of stool studies in all patients with abdominal pain and hematochezia, even with a pre-existing diagnosis of IBD.Figure 1

The Vast and Varied Global Burden of Norovirus: Prospects for Prevention and Control.

Globally, norovirus is associated with approximately one-fifth of all diarrhea cases, with similar prevalence in both children and adults, and is estimated to cause over 200,000 deaths annually in developing countries. Norovirus is an important pathogen in a number of high-priority domains: it is the most common cause of diarrheal episodes globally, the principal cause of foodborne disease outbreaks in the United States, a key health care–acquired infection, a common cause of travel-associated diarrhea, and a bane for deployed military troops. Partly as a result of this ubiquity and burden across a range of different populations, identifying target groups and strategies for intervention has been challenging. And, on top of the breadth of this public health problem, there remain important gaps in scientific knowledge regarding norovirus, especially with respect to disease in low-income settings.Many pathogens can cause acute gastroenteritis. Historically, rotavirus was the most common cause of severe disease in young children globally. Now, vaccines are available for rotavirus and are universally recommended by the World Health Organization. In countries with effective rotavirus vaccination programs, disease due to that pathogen has decreased markedly, but norovirus persists and is now the most common cause of pediatric gastroenteritis requiring medical attention. However, the data supporting the precise role of norovirus in low- and middle-income settings are sparse. With vaccines in the pipeline, addressing these and other important knowledge gaps is increasingly pressing.We assembled an expert group to assess the evidence for the global burden of norovirus and to consider the prospects for norovirus vaccine development. The group assessed the evidence in the areas of burden of disease, epidemiology, diagnostics, disease attribution, acquired immunity, and innate susceptibility, and the group considered how to bring norovirus vaccines from their current state of development to a viable product that will benefit global health.

Rapid, simple and efficient method for detection of viral genomes on raspberries

In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method allowed their recovery. Direct RNA extraction proved to be more effective (best recovery rate), faster (<8h) and simpler (fewer steps) than the one proposed in the CEN ISO standard method when it came to detecting enteric viruses on raspberries.

Citrobacter braakii: A Major Cause of False‐Positive Results on MacConkey and Levine's Eosin Methylene Blue Selective Agars Used for the Isolation of Escherichia Coli from Fresh Vegetable Samples

AbstractFresh vegetables are a frequent cause of foodborne disease outbreaks because they often do not undergo heat treatment prior to consumption. Pathogenic Escherichia coli strains are the major causative agents of produce‐related outbreaks. In this study, we investigated the efficiency of conventional E. coli selective medium, MacConkey (MAC) agar, for the screening of fresh vegetable samples and evaluated the discrimination ability of Levine's eosin methylene blue (L‐EMB) agar for E. coli‐like colonies obtained from MAC agar. A total of 120 samples of lettuces (n = 60) and radish sprouts (n = 60) were analyzed. Among 17 (14.2%) MAC agar plates containing putative E. coli colonies, only one plate was confirmed E. coli‐positive (positive predictive value = 5.9%). All 16 false‐positive isolates were identified as Citrobacter braakii and formed E. coli‐like colonies on L‐EMB agar. These results indicate that in order to reduce false‐positive results in screening fresh vegetable samples, both MAC and L‐EMB selective agars should be modified with the aim of providing reliable differentiation between E. coli and C. braakii.Practical ApplicationsAlthough MacConkey (MAC) and Levine's eosin methylene blue (L‐EMB) agars are not specific for the detection of pathogenic Escherichia coli, the media are recommended for the isolation of pathogenic E. coli (except E. coli O157:H7) in the Food and Drug Administration Bacteriological Analytical Manual. This is due to the fact that it is difficult to develop each pathogenic strains‐specific selective media as they were not classified by their biochemical characteristics. However, the unexpected high prevalence of Citrobacter braakii in fresh vegetables observed in this study indicates that the screening potential of these E. coli selective media needs to be improved.

Transfer of Acanthamoeba spp. to fresh produce from water and environmental surfaces.

Human noroviruses (HuNoV) are the primary cause of food-borne disease outbreaks in the United States. The most frequent commodities implicated in HuNoV outbreaks are leafy greens where contamination may occur during production and harvesting practices. With respect to the transmission of HuNoV to fresh produce, one hypothesis is that free-living amoebae that are ubiquitous in the environment (soil, sediments and water) can serve as vehicles of contamination through interaction with viruses. Here, we investigated the transfer of Acanthamoeba spp. both alone and associated with murine norovirus (MNV-1)-a surrogate for HuNoV-from water and food contact surfaces to fresh produce to understand the transfer of amoebae and the effect of virus association with amoeba on transferability, if any. In water containing a low concentration of amoebae (3log10 cellml(-1) ), 3·85log10 amoebae transferred to 5g of leafy greens, and for 5 cherry tomatoes, 3·4 to 3·5log10 amoebae were transferred. Similarly, for high concentrations of amoeba (5log10 cellml(-1) ) in water, 6·14 and 5·81log10 amoebae were transferred to 5g leafy greens and five cherry tomatoes respectively. However, the transfer of amoebae from food contact surfaces to fresh produce was very limited. In addition, amoebae association with MNV-1 did not impact transferability. The results of this study provide a better understanding of physical parameters (e.g. surface area and texture of fresh produce, transfer medium-water vs surface) potentially associated with transfer of free-living amoeba to fresh produce as well as the role that contaminated water (irrigation or wash water) may play in the transmission of enteric viruses associated with amoeba.

Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)-ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce-could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce.

Survival of hepatitis A virus on various food-contact surfaces during 28 days of storage at room temperature

Hepatitis A virus (HAV) infections constitute one of the leading causes of food-borne disease outbreaks. HAV spreads readily from one infected person to another through contaminated water or food, via the fecal–oral route. The prevention of HAV infection is therefore important in the preparation of food. The objective of this study was to investigate the survival of HAV on six different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel and plastic. The survival of HAV was measured during storage at room temperature for 28days. On the food-contact surfaces, the greatest reduction in HAV was 2.3log10plaque-forming units (PFU)/coupon, observed on stainless steel, while the lowest HAV reduction was 1.4log10PFU/coupon, observed on wood. The values of dR (time required to reduce the virus by 99%) on survival plots of HAV determined by a modified Weibull model were 1396.9h (R2=0.97) on ceramic, 1676.4h (R2=0.98) on wood, 852.7h on rubber (R2=0.95), 1386.4h (R2=0.97) on glass, 428.6h (R2=0.96) on stainless steel and 833.0h (R2=0.97) on plastic. The infectivity of the virus on all six food-contact surfaces was maintained after 28days. Studies show that HAV from food preparation or processing will survive persistently on cookware. This study provides information useful for the control of HAV on different food-contact surfaces and the prevention of food-borne disease.

Sources of Calicivirus contamination in foodborne outbreaks in Denmark, 2005-2011--the role of the asymptomatic food handler.

Norovirus (NoV) is the predominant cause of foodborne disease outbreaks. Virus contamination may occur during all steps of food processing, from production to preparation and serving. The relative importance of these different routes of contamination is unknown. The purpose of this study was to estimate the proportions of outbreaks caused by asymptomatic and symptomatic food handlers (FHs). Reports of foodborne NoV and sapovirus outbreaks (n=191) that occurred over a 7-year period were extracted, reviewed, and categorized according to the available evidence for source of contamination. In 64 (34%) of the outbreaks, contamination from FHs took place during preparation or serving of food. In the majority of these outbreaks (n=41; 64%), the FHs were asymptomatic during food handling. Some had been in contact with ill household members before handling the food and remained asymptomatic; others developed symptoms shortly after or were post-symptomatic. In 51 (27%) of the outbreaks, contamination occurred during production of the food, and in 55 (29%) of the outbreaks, contamination had supposedly occurred after serving a guest at a self-serve buffet. Guidelines regarding exclusion of FHs where household members suffer from gastroenteritis could limit the number of outbreaks.