In vitro antimicrobial activity of extracts and essential oils of Cinnamomum, Salvia, and Menthaspp. against foodborne pathogens: a meta‐analysis study

Genetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.Contributions of Jakob Wegener and Eduard Musin were supported by funds of the Federal Ministry of Food and Agriculture based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food, grant number 2818BM040. Dora Henriques was supported by BEEHAPPY (POCI-01-0145- FEDER-029871; FCT and COMPETE/QREN/EU). Ana R. Lopes and Carlos A. Yadró were supported by Fundaçãoo para a Ciência e a Tecnologia (FCT) grants SFRH/BD/143627/2019 and 2021.06948.BD, respectively. FCT provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021).info:eu-repo/semantics/publishedVersio

An Environmental Approach To Food Safety Assessment Using Artisan Cheese And Fresh Produce As Model Systems

This dissertation examined recently issued regulatory standards using cheese and produce as model systems. FDA\u27s 2015 Domestic and Imported Cheese and Cheese Products Compliance Program Guidelines (CPG) E. coli standards on cheese safety, and the extent to which these standards affect domestic and imported cheese commerce, was assessed. Results from FDA\u27s Domestic and Imported Cheese Compliance Program for samples collected between January 1, 2004 and December 31, 2006 were analyzed. Of 3,007 cheese samples tested for non-toxigenic E. coli, 76% (2,300) of samples exceeded 10/g, FDA\u27s target for regulatory activity. In cheese samples containing E. coli levels of 10/g and 100/g, there was no statistically significant association with presence of Listeria monocytogenes. However, associations between Staphylococcus aureus levels of 10,000 CFU/g and presence of Salmonella and L. monocytogenes were statistically significant, indicating that EU regulations targeting S. aureus as the pathogen of concern may be more appropriate than E. coli for cheese safety assessment. Compost amended soils in the Northeastern U.S. were assessed for the presence and survival of E. coli and Listeria spp. against FDA Food Safety Modernization Act (FSMA) requirements. Manure and poultry litter-based biological soil amendments of animal origin (BSAAO) must achieve pathogen reduction to reduce risk of pathogen contamination on the harvested produce. Two trials of replicated field plots of loamy (L) or sandy (S) soils were tilled and un-amended (NC) or amended with dairy compost (DC), poultry litter compost (PL), or poultry pellets (PP). Colony count and most probable number (MPN) methods were used to determine persistence of E. coli in these plots over 104 days post-inoculation. Detection of indigenous Listeria spp. were also examined in all plots. Higher E. coli populations were observed in PL and PP amended soils when compared to DC and NC plots. E. coli was detected at low levels on radish crops, where PL treatments encouraged greater levels of survival and growth than DC or NC. Study results verify that a 120 day interval following BSAAO application should be sufficient to ensure food safety of edible crops subsequently planted on these soils. The sensitivity of environmental monitoring methods and collection formats were evaluated to identify optimal procedures for detection of Listeria spp. on product contact surfaces within artisan cheese production environments. Four environmental surfaces (dairy brick, stainless steel, plastic, and wood; n=405/surface type) were inoculated with L. innocua, L.m. ATTC® 19115 and L.m. 1042, at high (106-107/cm2) and low (0.1-1/cm2) target concentrations. Inoculated surfaces were swabbed with World Bioproducts© EZ ReachTM and 3MTM environmental swabs (3MTM). Five enrichment and enumeration methods were used to compare sensitivity of recovery between environmental swabs. All swab formats performed equally on all environmental surfaces at high target concentrations. At low concentrations, PetrifilmTM and WBEZ swabs recovered Listeria spp. from 87.5% of plastic, stainless steel, and dairy brick surfaces, but only 62.7% of wooden surfaces; recovering 14.8%, 77%, and 96.3% of cells from initial inoculations of 0.01, 0.1, and 1/cm2, respectively . Our data demonstrate that results may be discrepant due to variation in the porosity of environmental surfaces and should be taken into consideration when implementing environmental sampling plans. Results from this thesis can be used to inform regulatory policy and help to achieve improved food safety

THE SURVIVAL OF VARIOUS PATHOGENIC ORGANISMS IN FATS AND OILS

The research within this thesis sought to determine the ability of various animal derived fats and plant derived oils to support the survival of several pathogenic cocktails over a multitude of storage times. The Salmonella study explored the survival rate of a four strain Salmonella cocktail in beef tallow, pig lard, duck fat, coconut oil, and extra virgin olive oil over seven days at 26˚C and 37˚C storage. The animal fats and the coconut oil supported the survival of the bacteria until the conclusion of the study. The Shiga-toxin producing Escherichia coli study explored the survival rate of a five strain STECs cocktail in extra virgin olive oil over seven days at 26˚C and 37˚C storage. The two Listeria studies explored the survival rate of a four strain Listeria monocytogenes cocktail in extra virgin olive oil over several time periods with different frequencies of sample mixing. In vitro, all genuses showed a 2.5-log cfu/mL to ≥ 7-log cfu/mL reduction in the extra virgin olive oil by the conclusion of the experiments. Extra virgin olive oil was then applied to cooked pork tenderloin, cheddar cheese snack squares, and turkey lunchmeat in hopes of inhibiting the L. monocytogenes cocktail. No reduction was observed

Prevalence, behavior and risk assessment of Salmonella spp. and Shiga toxin-producing Escherichia coli on basil leaves and strawberries

Consumption of fresh fruits and vegetables (F&V) is important for a healthy and balanced diet due to the presence of vitamins, minerals, fibres and other dietary components. However, F&V, in particular leafy green vegetables, that are consumed raw, have been associated with some notable outbreaks of microbial foodborne disease. Within this context and due the lack of data relating to the prevalence and behaviour of pathogens on F&V, the EU FP7 Veg-i-Trade project was developed. This PhD research deals with the microbiological analysis and exposure assessment of particular case studies of F&V being Salmonella and STEC in strawberries and basil. The objective of the PhD research was (i) to collect information about the prevalence of Salmonella and STEC on basil and strawberries and to investigate the behaviour of these pathogens during the storage of these commodities at different reasonably foreseen temperatures and (ii) subsequently use the obtained data as input in the comparative exposure assessment model for a quantitative risk ranking. To do so, first detection methods (i.e. the GeneDisc multiplex real-time PCR kit and culture methods for detection of Salmonella and STEC) needed some evaluation for their appropriateness in use for these specific food products (iii). In addition, exposure to pathogens due to consumption of basil and strawberries were compared to exposure to already prior well studied lettuce as a leafy green

Uptake of enterohemorrhagic Escherichia coli into the roots of lettuce plants

Within the last 10 years, the annual numbers of human infections with enterohemorrhagic Escherichia coli (EHEC) in Germany increased by a factor of 2.4. The peak was reached during the large German outbreak in 2011. Intriguingly, the source of the outbreak was supposedly traced back to organic fenugreek sprouts. Moreover, the number of EHEC outbreaks traced back to plant-based foods, e.g. fresh produce, increased also in the United States. This trend poses a serious threat to public health as fresh produce is mostly consumed raw. Also, these observations gave rise to investigate the interactions of plants and human pathogens in more detail especially as fresh produce may be contaminated directly on the field. In the present thesis, the capability of different EHEC strains and an enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strain, to adhere to and to internalize into the roots of different lettuce plants was investigated. These studies conducted within the scope of this dissertation focused on different aspects of the mentioned processes, such as different bacterial strains, the bacterial genetic equipment, and different environmental conditions, such as plant variety, soil type used for plant growth, and the soil microbiota. To mimic the natural conditions as close as possible, plants were grown from unsterile plant seed in unsterile soil under greenhouse conditions. In the first publication, the overall ability of EHEC O157:H7 strain Sakai to adhere to and internalize into the roots of Valerianella locusta, also known as lambs lettuce, grown in diluvial sand soil was described. It was demonstrated that EHEC O157:H7 strain Sakai is indeed able to attach to and internalized into the lettuce roots under the conditions tested. Moreover, this paper shed light on potentially important intrinsic bacterial factors, i.e. genes/proteins, which are putatively involved in adherence and/or internalization. Therefore, deletion mutants lacking hcpA and/or iha, were also investigated regarding adherence to and internalization into the lambs lettuce roots. Both genes, coding for the major subunit of the hemorrhagic coli pilus HcpA and the adhesin Iha, respectively, are supposed to be associated with adherence and therefore called adherence factors. However, deletion mutants lacking one or both of these genes did not show significant differences in root attachment compared to the wild-type strain. Regarding internalization, deletion of either of these genes resulted in significantly lower numbers of internalized bacteria clearly indicating that both of these genes or the proteins encoded by these genes play an important role during invasion of EHEC O157:H7 strain Sakai into the roots of lambs lettuce. Interestingly, deletion of both genes did not result in further reduction of internalization compared to single deletion mutants. Hence, hcpA and iha encode rather internalization factors than adherence factors. Moreover, internalization does not solely depend on these two factors. The second paper focused on the influence of lettuce varieties and soil type on adherence and internalization of E. coli O104:H4 strain C227/11φcu. In this study, the lettuce varieties Valerianella locusta and Lactuca sativa, also known as lambs lettuce and lettuce, respectively, were both grown in two different soil types, diluvial sand (DS) and alluvial loam (AL), to address the impact of plant host and environment on bacterial attachment and invasion into lettuce roots. To approach the latter aspect in more detail, the composition of the soil microbial community was analyzed in parallel by partial 16S rRNA gene sequencing. Adherence to the roots was positively influenced by the soil type as the number of adherent E. coli O104:H4 strain C227/11φcu bacteria significantly rose by a factor of three to four when the plants were grown in DS compared to AL. However, when grown in the same type of soil, no statistically significant differences in attachment were detected between the distinct lettuce varieties. On the other hand, internalization significantly differed predominantly between the two types of lettuce. Internalization into the roots of L. sativa compared V. locusta was found to be increased by a factor of 12 upon growth in DS, and by a factor of 108 when the plants were grown in AL. Moreover, internalization into the roots of L. sativa was five-times higher in AL than in DS. Consequently, the lettuce variety significantly influences to ability of E. coli O104:H4 strain C227/11φcu to internalize into the lettuce roots, while the soil type affected bacterial invasion only at the roots of L. sativa under the conditions tested. Moreover, by microbiota analysis, the inoculated strain was found within the soil microbiota, and this analysis demonstrated that soil type, lettuce variety, and the combination of both result in large differences in the composition of the soil microbiota.In den letzten 10 Jahren hat sich die jährliche Fallzahl der Infektionen mit enterohämorrhagischen Escherichia coli (EHEC) in Deutschland mehr als verdoppelt. Den Höhepunkt der EHEC-Erkrankungen in Deutschland stellte der große Ausbruch von 2011 dar. Interessanterweise waren wahrscheinlich Bio-Bockshornkleesprossen die Infektionsquelle. Zusätzlich steigt auch in den Vereinigten Staaten die Anzahl der EHEC-Ausbrüche, die auf pflanzliche Lebensmittel, wie etwa Salat, zurückzuführen sind. Diese Entwicklung ist eine ernstzunehmende Gefahr für die öffentliche Gesundheit, da Frischwaren wie Salat meist roh verzehrt werden. Diese Beobachtungen gaben außerdem Anlass die Interaktion von Pflanzen und Humanpathogenen genauer zu untersuchen, vor allem da Frischwaren direkt auf dem Feld kontaminiert werden können. In dieser Arbeit wurden verschiedene EHEC-Stämme und ein enteroaggregativer/enterohämorrhagischer E.coli (EAEC/EHEC) Stamm, untersucht, hinsichtlich ihrer Fähigkeit an die Wurzeln unterschiedlicher Salatsorten zu adhärieren und in diese zu internalisieren. Die Studien, die im Rahmen dieser Dissertation durchgeführt wurden, fokussierten sich dabei auf unterschiedliche Aspekte dieser Prozesse, wie etwa unterschiedliche Bakterienstämme, genetische Ausstattung der Bakterien, und verschiedene Umweltbedingungen, wie Salatsorte, für die Anzucht verwendeter Bodentyp und die Bodenmikrobiota. Um die natürlichen Bedingungen möglichst genau nachzuahmen, wurden die Pflanzen aus nicht sterilem Saatgut in nicht sterilem Boden in einem Gewächshaus angepflanzt. Die erste Publikation befasste sich mit der prinzipiellen Fähigkeit EHEC O157:H7 Stamm Sakai an die Wurzeln von Valerianella locusta, Feldsalat, zu adhärieren und in diese zu internalisieren, wobei der Salat in Diluvialsand gewachsen ist. Es wurde gezeigt, dass EHEC O157:H7 Stamm Sakai unter den getesteten Bedingungen adhärieren und internalisieren kann. Außerdem wurde die Rolle von potentiell wichtigen intrinsischen Bakterienfaktoren untersucht, d.h. Gene bzw. Proteine, die potenziell in Adhärenz und/oder Internalisierung involviert sind. Dazu wurden hcpA- und/oder iha-negative Deletionsmutanten generiert. Diese wurden ebenso bezüglich Adhärenz und Internalisierung an bzw. in die Wurzeln von Feldsalat untersucht. Die beiden Gene kodieren die Hauptuntereinheit des hämorrhagischen Coli Pilus bzw. das Adhesin Iha und werden Adhärenzfaktoren genannt, da sie im Zusammenhang mit Adhärenz stehen sollen. Jedoch zeigten weder die beiden Einzel- noch die Doppelmutante signifikante Veränderungen im Adhärenzverhalten im Vergleich zum Wildtyp. Bezüglich der Internalisierung führte die Deletion eines der beiden Gene zu einer signifikant geringeren Zahl von internalisierten Bakterien, was darauf schließen lässt, dass diese Gene oder die codierten Proteine eine wichtige Rolle spielen bei dem Eindringen von EHEC O157:H7 Stamm Sakai in die Wurzeln von Feldsalat. Jedoch zeigte die Doppelmutante keine weitere Reduktion der Internalisierung verglichen mit den Einzelmutanten. Ergo kodieren hcpA und iha eher Internalisierungfaktoren als Adhärenzfaktoren. Außerdem hängt Internalisierung nicht ausschließlich von diesen beiden Faktoren ab. Die zweite Publikation fokussierte sich auf den Einfluss von Salatsorte und Bodentyp auf Adhärenz und Internalisierung von E. coli O104:H4 Stamm C227/11φcu. In dieser Studie wurden die beiden Salatsorten Valerianella locusta, Feldsalat, und Lactuca sativa, Kopfsalat, in verschiedenen Bodentypen, Diluvialsand (DS) und Alluviallehm (AL) angepflanzt, um Auswirkungen von Pflanzenwirt und Umwelt auf Anheftung und Eindringen der Bakterien zu untersuchen. Um den Umweltaspekt genauer zu beleuchten, wurde parallel die Komposition der Bodenmikrobiota mittels partieller 16S rRNA Gensequenzierung untersucht. Die Adhärenz an die Wurzeln wurde positiv vom Bodentyp beeinflusst. Die Zahl der adhärenten E. coli O104:H4 Stamm C227/11φcu stieg um den Faktor drei bis vier bei Pflanzenwachstum in DS im Vergleich zu AL. Bei gleichem Bodentyp zeigten sich keine signifikanten Unterschiede zwischen den Salatsorten. Die Internalisierung jedoch unterschied sich vor allem zwischen den Salatsorten. Bei Pflanzenwachstum in DS wurde in den Kopfsalatwurzeln das 12-fache an internalisierten Bakterien gefunden im Vergleich zu Feldsalatwurzeln. Bei Wachstum in AL war es gar das 108-fache. Zudem war die Internalisierung in die Wurzeln von L. sativa in AL fünfmal höher als in DS. Die Salatsorte hat somit einen signifikanten Einfluss auf die Internalisierungsfähigkeit von E. coli O104:H4 Stamm C227/11φcu. Der Bodentyp beeinflusste unter den getesteten Bedingungen aber nur das Eindringen in Kopfsalatwurzeln. Außerdem konnte der inokulierte Bakterienstamm mittels Mikrobiotaanalyse innerhalb der Bodenmikrobiota detektiert werden. Diese Analyse zeigte, dass sowohl Bodentyp als auch Salatsorte sowie die Kombination von beiden zu großen Unterschieden in der Komposition der Bodenmikrobiota führen

Thermal Inactivation of Shiga Toxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e in Foods

Emerging non-O157 Shiga toxin-producing Escherichia coli (STEC) were recently added to the zero tolerance policy by the USDA-FSIS. Therefore, the precise characterization of their thermal inactivation kinetics in different foods and the effect of stress on thermal inactivation are needed. This research aimed at determining the heat inactivation kinetics of non-O157 and O157 STECs in buffer and model food matrices and the effects of DnaK levels on thermal resistance after acid and heat-shock. Thermal inactivation was carried out in either in 2-ml glass vials or nylon vacuum-sealed bags for buffer and food (spinach, ground-beef, turkey deli-meat, pasta) samples, respectively. Vials or bags were immersed in a re-circulating water bath at various set temperatures for fixed time-intervals. Surviving bacteria were enumerated using Tryptic Soy Agar plates. D-values were calculated using first-order linear and Weibull (for pasta only) models. Total bacterial protein (using Bicinchonic acid assay) and the heat-shock protein (DnaK) concentration were measured (using competitive ELISA) before and after treating overnight-grown cells in Tryptic Soy Broth with either acid- (acetic acid pH 5.5 for 1 h) or heat-shock (46°C for 15 min). All experiments were performed in duplicate and replicated thrice, data were analyzed using SAS (pE. coli O157 and non-O157 in all tested samples, at 56°C D-values ranged from 5.57±0.38 to 15.39±0.14 min; at 58°C D -values ranged from 1.99±0.9 to 7.20±0.55 min and at 60°C D–values ranged from 0.99±0.07 to 2.86±0.22 min. Higher levels of DnaK were detected after sub-lethal injury with heat- or acid-shock that corresponded to enhanced thermal tolerance of all strains, except E. coli O111. D-values in buffer for un-shocked cells ranged from 1.49±0.35 to 2.21±0.17 min, heat-shocked cells from 2.04±0.35 to 2.83±0.35 min and acid-shocked cells from 2.32±0.29 to 4.09±0.29 min. Thus, acid- or heat-shock conditions that might occur during food processing need to be considered during design of thermal inactivation processes and product-formulation to prevent food-borne outbreaks. This study provides insights on the thermal inactivation parameters of O157 and non-O157 STEC in foods that would be beneficial to the food industry

Microbiological hygiene and biological control of leafy green vegetables

Fruit and vegetables are vital components of a healthy diet, and international strategies to encourage their consumption are in place (FAO/WHO, 2005). Ready-to-eat (RTE) leafy green vegetable products have exploded in popularity, and are a convenient and attractive way to add greens to the plate. Unfortunately, sales numbers are accompanied by increasing numbers of food-borne illness outbreaks.This thesis focuses on the microbial hygiene from a consumer’s perspective, followed by application and evaluation of biological control as a mean of reducing the risk of food-borne illness. When studying the efficacy of two different household washing methods to reduce the bacterial load on leafy green vegetables, it was seen that only after rinsing at high water velocity (8 L/min), after five repetitions, the bacterial load decreased with 90 %. The treatment disintegrated the leaves, and still left the produce with high amounts of culturable bacteria. These results highlight the inefficiency of tap water washing methods available for the consumer. Consumer habits are also important to consider when assessing the microbial hygiene of food products. Packages of RTE leafy green products were opened, stored at 7 ˚C and compared with unopened bags. The total aerobic count from different producers varied greatly and no correlation to opened bags could be made. Neither could bacterial levels be linked to a certain producer or product type.Inoculation with E. coli strains indicate that the type strain is able to survive, but not multiply, in household conditions. However, wild strains of E. coli were seen to multiply at different time-points during the shelf-life period, adapting to cold storage conditions. This varying and unpredictable bacterial status of ready to eat leafy green products calls for new strategies to reduce unwanted microorganisms and prevent food-borne illness.By the means of biological control, bacteria can be used to counteract food safety hazards. Therefore, isolates antagonistic to E. coli have been isolated and evaluated in an industrial field production setting. Selected isolates showing antagonism in vitro were coated onto spinach seeds before planting. Next generation sequencing analysis revealed that the microbiota of the plants inoculated with the selected strains was altered in a beneficial direction, and a reduction of Escherichia-Shigella could be seen during the development from seed to plant.As a tentative safety evaluation of the selected strains for biological control, an individual comparison for immunomodulating effects in mice was made. The two Bacillus coagulans strains consistently resemble the response of untreated animals, which must be considered a positive trait. The strain of Pseudomonas punonensis had a weaker influence on the immune system, while the Pseudomonas cedrina strain and the Rhocococcus cerastii strain induced inflammatory responses. The P. punonensis strain and one B. coagulans strain increased the microbiota diversity, which is correlated to host health.These results encourage the usage of bacterial antagonists as part of the solution to reduce the risk of human pathogens on leafy green vegetables

Development of a Bacteriophage-based Portable Biosensor for the Detection of Shiga-toxin Producing Escherichia coli (STEC) Strains in Food and Environmental Matrices

A fast and reliable on-site foodborne pathogens screening can reduce the incidence of foodborne illnesses, hospitalizations and economic loss. It can also circumvent conventional laboratory-based tests with minimal sample treatments and shorter turnaround time. Rapid detection of biological hazards has been largely dependent on immunological agents (ie antibodies). Antibodies are expensive to manufacture and experience cross-reactivity, instability with shorter shelf life. Our aim was to improve the screening process of Shiga-toxin producing Escherichia coli (STEC) strains in food and environmental matrices by developing a novel, inexpensive handheld bacteriophage-based amperometric biosensor that can directly detect live STEC cells.This biosensor development began by isolating STEC-specific bacteriophages from natural environmental samples (ie cow manure and surface water) hence, constructing a comprehensive bacteriophage isolates collection targeting an array of significant STEC serogroups. As an alternative to antibodies, purified bacteriophages could be easily and inexpensively propagated in a standard laboratory. Isolated bacteriophages were morphologically characterized while its physiologic behavior and specific host interactions were also investigated. The results indicated that majority of STEC-specific bacteriophages belong to Myoviridae and Siphoviridae families. Suitable bacteriophages for biosensor purposes were selected on the basis of the presence of head and tail and absence of virulence genes (stx1/stx2). Chemical modification via site-specific biotinylation of bacteriophage heads was performed prior to its biosensor incorporation. The results showed that biotinylation of bacteriophages did not reduce biofunctionality. Representative STEC O26, O157, and O179-specific biotinylated bacteriophages were immobilized onto the surface of streptavidin-modified screen-printed carbon electrodes (SPCE) to capture their target STEC cells. After STEC cells were bound to the capture elements, another set of biotinylated bacteriophages labeled with streptavidivin-horseradish peroxidase were added forming stable binding complexes which were then subjected to amperometric detection. The sandwich-type bacteriophage-based detection approach allowed live STEC cells rapid detection in microvolume samples (50 µL) via amperometric readouts (∆ current) between target and non-target bacteria in pure culture setup and complex matrices. With its simplicity and reliability, this technology can immensely assist the food industry and regulatory inspectors to efficiently maintain food safety in a fraction of the cost of traditional method

Antimicrobial efficacy of commercial produce sanitizers against artificially inoculated foodborne pathogens and natural fungal contaminants on the surface of whole melons

Foodborne disease outbreaks linked to enteric pathogens on cantaloupe and watermelon over the past few years has raised concern in the melon industry. As a result, new formulations of antimicrobial treatments have been developed, creating more choices for melon producers to utilize in attempts to improve microbial safety of their product. Previous research has indicated that the use of these strong post-harvest sanitizers may have detrimental quality effects on produce. The overall objective of this research was to evaluate the broad spectrum antimicrobial activity of commercially available produce sanitizers marketed towards the melon industry. Sanitizers selected represented various chemical categories of sodium hypochlorite (chlorine), hydrogen peroxide, liquid chlorine dioxide, organic acids, quaternary ammonium, and hydrogen peroxide/acid combinations. Bacterial reduction of E. coli O157:H7, non-O157 STEC, Listeria monocytogenes, and Salmonella spp. following in- cell suspension treatment (5 minutes, 4°C) with the ten different commercial sanitizers was evaluated at varying concentrations. Subsequently, selected sanitizers and concentrations were evaluated for their effectiveness in reducing the pathogen populations on the surface of artificially inoculated whole cantaloupe and watermelon rinds at representative packing house conditions of 24°C for 2 minute application times. Sanitizers displaying the greatest antimicrobial activity against pathogens and on both melon types were selected for evaluation of antifungal (yeast, mold) activities as well as quality effects over the shelf life of whole cantaloupe and watermelon following similar treatment conditions. Additional work using UV spectrometry and viable pathogen reduction was completed to investigate the antibacterial mechanism of action these produce sanitizers may utilize. Results of the cell suspension study revealed ability of all tested sanitizers to reduce pathogens by 0.6-9.6 log CFU/ml. One organic acid (citric/lactic acid combination) treatment resulted in minimal reduction (3 log or less) of all pathogens and was eliminated for further analysis. The application of sanitizers on melon rinds resulted in significant differences in pathogen reduction observed between sanitizers, but not between melon types. The most effective sanitizers against pathogens on melon surfaces were quaternary ammonium and hydrogen peroxide/acid combinations with 1.0-2.2 log CFU/gm and 1.3-3.5 log CFU/gm reductions, respectively, for all pathogens tested. The other tested sanitizers were less consistent with microbial kill, with reductions ranging from 0-3 log CFU/gm depending on pathogen and sanitizer. Results of the quality analysis following treatment with quaternary ammonium and hydrogen peroxide/acid combinations indicated no reduction of natural yeast and mold contaminants, and no changes in firmness or color of melon rinds (P\u3e0.05). Minimum bacterial concentration (MBC) of sanitizers against E. coli O157:H7 and L. innocua were determined, and varied by active ingredient. Mechanism of action analysis indicated that cell viability of E. coli O157:H7, non-O157 STEC, L. innocua, and Salmonella spp. decreased with time following treatment with all chemical sanitizers, with the exception of sodium hypochlorite. An increase in cellular leakage of A260 material was found to result in cells treated with quaternary ammonium, but not with any other tested sanitizer. Based on the results of the present work it can be concluded that 1) the use of broad spectrum produce sanitizers at recommended concentrations and treatment applications on the rind surfaces of whole melons has the potential to reduce bacterial foodborne pathogens of concern in the melon industry, 2) the use of commercial produce sanitizers may not reduce the presence of natural fungal contaminants on melon rinds but will not negatively affect the sensory quality of melons, and 3) death of bacterial cells exposed to quaternary ammonium products involves damage to cytoplasmic membrane but the mechanism of action utilized by additional tested produce sanitizers may involve more complex cellular interactions in addition to membrane damage. Overall, results of this research will be utilized to provide guidance to the melon industry on best produce sanitizers for a broad spectrum pathogen intervention strategy

Natural Product Heme Oxygenase Inducers as Treatment for Nonalcoholic Fatty Liver Disease

Heme oxygenase (HO) is a critical component of the defense mechanism to a wide variety of cellular stressors. HO induction affords cellular protection through the breakdown of toxic heme into metabolites, helping preserve cellular integrity. Nonalcoholic fatty liver disease (NAFLD) is a pathological condition by which the liver accumulates fat. The incidence of NAFLD has reached all-time high levels driven primarily by the obesity epidemic. NALFD can progress to nonalcoholic steatohepatitis (NASH), advancing further to liver cirrhosis or cancer. NAFLD is also a contributing factor to cardiovascular and metabolic diseases. There are currently no drugs to specifically treat NAFLD, with most treatments focused on lifestyle modifications. One emerging area for NAFLD treatment is the use of dietary supplements such as curcumin, pomegranate seed oil, milk thistle oil, cold-pressed Nigella Satvia oil, and resveratrol, among others. Recent studies have demonstrated that several of these natural dietary supplements attenuate hepatic lipid accumulation and fibrosis in NAFLD animal models. The beneficial actions of several of these compounds are associated with the induction of heme oxygenase-1 (HO-1). Thus, targeting HO-1 through dietary-supplements may be a useful therapeutic for NAFLD either alone or with lifestyle modifications