Sarah M. Markland

Ozone inactivation of norovirus surrogates on fresh produce.

Preharvest contamination of produce by foodborne viruses can occur through a variety of agents, including animal feces/manures, soil, irrigation water, animals, and human handling. Problems of contamination are magnified by potential countrywide distribution. Postharvest processing of produce can involve spraying, washing, or immersion into water with disinfectants; however, disinfectants, including chlorine, have varying effects on viruses and harmful by-products pose a concern. The use of ozone as a disinfectant in produce washes has shown great promise for bacterial pathogens, but limited research exists on its efficacy on viruses. This study compares ozone inactivation of human norovirus surrogates (feline calicivirus [FCV] and murine norovirus [MNV]) on produce (green onions and lettuce) and in sterile water. Green onions and lettuce inoculated with FCV or MNV were treated with ozone (6.25 ppm) for 0.5- to 10-min time intervals. Infectivity was determined by 50% tissue culture infectious dose (TCID(50)) and plaque assay for FCV and MNV, respectively. After 5 min of ozone treatment, >6 log TCID(50)/ml of FCV was inactivated in water and ∼2-log TCID(50)/ml on lettuce and green onions. MNV inoculated onto green onions and lettuce showed a >2-log reduction after 1 min of ozone treatment. The food matrix played the largest role in protection against ozone inactivation. These results indicate that ozone is an alternative method to reduce viral contamination on the surface of fresh produce.

Survival of Murine Norovirus, Tulane Virus, and Hepatitis A Virus on Alfalfa Seeds and Sprouts during Storage and Germination

ABSTRACT Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID50)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID50/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.

Pathogenic Psychrotolerant Sporeformers: An Emerging Challenge for Low-temperature Storage of Minimally-processed Foods

Sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. Beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. In these products, the sporeforming pathogens of concern are Bacillus cereus, Bacillus weihenstephanensis, and Clostridium botulinum type E. This review article examines the foods, conditions, and organisms responsible for the food safety issue caused by the germination and outgrowth of psychrotolerant sporeforming pathogens in minimally processed refrigerated foods.

Efficient reduction of pathogenic and spoilage microorganisms from apple cider by combining microfiltration with UV treatment.

Thermal pasteurization can achieve the U. S. Food and Drug Administration-required 5-log reduction of pathogenic Escherichia coli O157:H7 and Cryptosporidium parvum in apple juice and cider, but it can also negatively affect the nutritional and organoleptic properties of the treated products. In addition, thermal pasteurization is only marginally effective against the acidophilic, thermophilic, and spore-forming bacteria Alicyclobacillus spp., which is known to cause off-flavors in juice products. In this study, the efficiency of a combined microfiltration (MF) and UV process as a nonthermal treatment for the reduction of pathogenic and nonpathogenic E. coli, C. parvum, and Alicyclobacillus acidoterrestris from apple cider was investigated. MF was used to physically remove suspended solids and microorganisms from apple cider, thus enhancing the effectiveness of UV and allowing a lower UV dose to be used. MF, with ceramic membranes (pore sizes, 0.8 and 1.4 μm), was performed at a temperature of 10 °C and a transmembrane pressure of 155 kPa. The subsequent UV treatment was conducted using at a low UV dose of 1.75 mJ/cm(2). The combined MF and UV achieved more than a 5-log reduction of E. coli, C. parvum, and A. acidoterrestris. MF with the 0.8-μm pore size performed better than the 1.4-μm pore size on removal of E. coli and A. acidoterrestris. The developed nonthermal hurdle treatment has the potential to significantly reduce pathogens, as well as spores, yeasts, molds, and protozoa in apple cider, and thus help juice processors improve the safety and quality of their products.

Old friends in new places: exploring the role of extraintestinal E. coli in intestinal disease and foodborne illness.

The emergence of new antibiotic‐resistant Escherichia coli pathotypes associated with human disease has led to an investigation in terms of the origins of these pathogens. According to the Centers for Disease Control and Prevention, unspecified agents are responsible for 38.4 million of the 48 million (80%) cases of foodborne illnesses each year in the United States. It is hypothesized that environmental E. coli not typically associated with the ability to cause disease in humans could potentially be responsible for some of these cases. In order for an environmental E. coli isolate to have the ability to cause foodborne illness, it must be able to utilize the same attachment and virulence mechanisms utilized by other human pathogenic E. coli. Recent research has shown that many avian pathogenic E. coli (APEC) isolated from poultry harbour attachment and virulence genes also currently found in human pathogenic E. coli isolates. Research also suggests that, in addition to the ability to cause gastrointestinal illnesses, APEC may also be an etiological agent of foodborne urinary tract infections (FUTIs). The purpose of this article was to evaluate the evidence pertaining to the ability of APEC to cause disease in humans, their potential for zoonotic transfer along with discussion on the types of illnesses that may be associated with these pathogens.

Application of Bacillus subtilis to the roots of leafy greens, in the presence of Listeria innocua and Salmonella Newport, induces closure of stomata.

Plant growth-promoting rhizobacterium Bacillus subtilis UD1022 has been shown to trigger an induced systemic response in Arabidopsis thaliana. This interaction causes plant stomata to close, protecting the plant from infection by plant pathogens and thereby increasing crop yield. The purpose of this study was to determine whether UD1022 applied to the roots of plants is able to induce stomata closure in leafy greens as well as influence the persistence of human pathogens (Listeria and Salmonella) on plants. UD1022 induced stomata closure in the presence of human pathogens on both lettuce and spinach 3 h post-inoculation (p<0.0001). Results were confirmed by root inoculation with heat-killed UD1022, which did not induce stomata closure. Presence of UD1022 on lettuce roots significantly reduced the persistence of Listeria on plants after 3 days post-inoculation (p=0.02) but had less of an effect on the persistence of Salmonella. The results of this study indicate that plant growth-promoting rhizobacterium B. subtilis UD1022 may be able to prevent contamination by some human pathogens. This is the first study to investigate the use of plant growth-promoting rhizobacteria to control the persistence of human pathogens on plants.

An evaluation of the virulence and adherence properties of avian pathogenic Escherichia coli

Avian pathogenic E. coli (APEC) cause disease primarly in poultry; however, the link between APEC and infections in humans is questionable. In this current study, a total of 100 APEC strains isolated from chickens in Delmarva were evaluated for the presence of virulence genes to investigate their zoonotic potential in humans. A total of 28 isolates possessed one Enterohaemorrhagic E. coli (EHEC) virulence factor each and 87 isolates possessed up to 5 extraintestinal pathogenic E. coli (ExPEC) virulence factors. Five APEC isolates exhibited stronger attachment to chicken breast than both human E. coli outbreak strains tested. Ten APEC isolates exhibited stronger attachment to human epithelial cells (HCT-8) than both E. coli outbreak strains. While the APEC isolates in this study were not found to possess all the virulence genes necessary to cause clinical illness in humans, their potential to acquire these genes in the environment as well as their ability to attach to food surfaces and human cells warrants further attention.

Water for Agriculture: the Convergence of Sustainability and Safety

Agricultural water is a precious and limited resource. Increasingly more water types and sources are being explored for use in irrigation within the United States and across the globe. As outlined in this chapter, the Produce Safety Rule (PSR) in the Food Safety and Modernization Act (FSMA) provide irrigation water standards for application of water to fruits and vegetables consumed raw. These rules for production and use of water will continue to develop and be required as the world experiences aspects of a changing climate including flooding as well as drought conditions. Research continues to assess the use of agricultural water types. The increased use of reclaimed water in the United States as well as for selected irrigation water needs for specific crops may provide increased water availability. The use of surface water can be used in irrigation as well, but several studies have shown the presence of some enteric bacterial pathogens (enterohemorrhagic E. coli, Salmonella spp. and Listeria monocytogenes) in these waters that may contaminate fruits and vegetables. There have been outbreaks of foodborne illness in the U.S., South America, Europe, and Australia related to the use of contaminated water in fruit and vegetable irrigation or washing. Unreliable water supplies, more stringent microbial water standards, mitigation technologies and expanded uses of reclaimed waters have all increased interest in agricultural water.

RNA-seq: primary cells, cell lines and heat stress

Transcriptome analysis by RNA-seq has emerged as a high-throughput, cost-effective means to evaluate the expression pattern of genes in organisms. Unlike other methods, such as microarrays or quantitative PCR, RNA-seq is a target free method that permits analysis of essentially any RNA that can be amplified from a cell or tissue. At its most basic, RNA-seq can determine individual gene expression levels by counting the number of times a particular transcript was found in the sequence data. Transcript levels can be compared across multiple samples to identify differentially expressed genes and infer differences in biological states between the samples. We have used this approach to examine gene expression patterns in chicken and human cells, with particular interest in determining response to heat stress.

Inactivation of Human Norovirus and Its Surrogates on Alfalfa Seeds by Aqueous Ozone.

Alfalfa sprouts have been associated with numerous foodborne outbreaks. Previous studies investigated the effectiveness of aqueous ozone on bacterially contaminated seeds, yet little is known about the response of human norovirus (huNoV). This study assessed aqueous ozone for the disinfection of alfalfa seeds contaminated with huNoV and its surrogates. The inactivation of viruses without a food matrix was also investigated. Alfalfa seeds were inoculated with huNoV genogroup II, Tulane virus (TV), and murine norovirus (MNV); viruses alone or inoculated on seeds were treated in deionized water containing 6.25 ppm of aqueous ozone with agitation at 22°C for 0.5, 1, 5, 15, or 30 min. The data showed that aqueous ozone resulted in reductions of MNV and TV infectivity from 1.66 ± 1.11 to 5.60 ± 1.11 log PFU/g seeds; for all treatment times, significantly higher reductions were observed for MNV (P < 0.05). Viral genomes were relatively resistant, with a reduction of 1.50 ± 0.14 to 3.00 ± 0.14 log genomic copies/g seeds; the reduction of TV inoculated in water was similar to that of huNoV, whereas MNV had significantly greater reductions in genomic copies (P < 0.05). Similar trends were observed in ozone-treated viruses alone, with significantly higher levels of inactivation (P < 0.05), especially with reduced levels of infectivity for MNV and TV. The significant inactivation by aqueous ozone indicates that ozone may be a plausible substitute for chlorine as an alternative treatment for seeds. The behavior of TV was similar to that of huNoV, which makes it a promising surrogate for these types of scenarios.