Food Safety Masters Theses

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Antimicrobial Resistance and Presence of Integrons in Salmonella Isolated from Animals and Humans in the United States of America and Uganda
(North Dakota State University, 2010) Mahero, Michael Wandanje
Salmonella has been cited as one of the leading causes of food borne illness world wide and in the United States (US), as well as an indicator organism for studying antimicrobial resistance (AMR) trends. The objective of this study was to characterise AMR patterns of Salmonella isolates from animals and humans in North Dakota, US, and Kampala, Uganda, and determine the association between the observed AMR and presence of class 1 integrons. Salmonella isolates were collected from the Veterinary Diagnostic Laboratory (VDL) at North Dakota State University and the North Dakota Department of Health respectively from 2003-2008. Samples were also retrieved from archives present at the Microbiology Department, Faculty of Veterinary Medicine at Makerere University in Kampala, Uganda. AMR profiles were determined using a panel of 15 antimicrobials as per the manufacturer's instructions (Sensitire, Trek Diagnostics System, Westlake, Ohio). Screening for the class 1 integrons was done using PCR with primers specific for the int 1. Out of 359 Salmonella isolates tested, 24. 79% were resistant to ~5 antimicrobials while 36.2% were resistant to at least 2. Pan susceptible isolates were mostly (65.05%) from human isolates. The most common multidrug resistant (MDR) phenotype among the tested isolates was the classic ACSSuT penta-resistance at 29.06% (50/172). The highest resistance frequency was seen against Tetracycline (39.6%) and Streptomycin (34.7 %), while 5.2% (17) of the isolates were resistant to Nalidixic acid and 56 (15.7%) to Ceftiofur. A total of20.7% (57/276) of the ND samples tested positive for presence of class 1 integrons. Class 1 integron was significantly associated (p< 0.05) with AMR to Ampicillin, Kanamycin, Tetracycline, Streptomycin and Sulfisoxazole. Of all Ugai.dan Salmonella isolates tested, 94.4% (68/72) were resistant to 2':2 antimicrobials. The highest resistance was observed against Sulfisoxazole and Trimethoprim-Sulphamethoxazole, and 45.8% of human and 46.2% of cattle isolates tested positive for presence of class 1 integrons. Presence of class 1 integron was significantly associated (p< 0.05) with AMR to Tetracycline and Amoxicillin. DNA sequencing of the class 1 integron variable regions identified several resistance genes including aadAJ, dfrA 7, and dfrA5 gene. The data indicated high AMR among antimicrobials widely used in veterinary and human medicine. Also, AMR was observed against drugs whose veterinary use is restricted, implying possible horizontal transmission. A good proportion (47.9% in Uganda and 29.85% in ND) of the Salmonella isolates from clinical cases of salmonellosis were MDR (resistant to 2':2) isolates bearing class 1 integron.
Relatedness ofisolates of a Novel Genus, Cronobacter, Formerly Known as Enterobacter sakazakii
(North Dakota State University, 2010) Solseng, Tracy Anne
Members of the genus Cronobacter were once classified as Enterobacter sakazakii. These bacteria are opportunistic pathogens that are associated with necrotizing enteritis, sepsis and meningitis in neonatal or low-birth-weight infants and can result in death, slowed development, or extensive neurological disorders. In adults, they have been documented as a cause of bacteremia, osteomyelitis, and vaginitis. Previously, E. sakazakii was found in the midgut of stable flies. Research by Nangoh et al. determined that Cronobacter spp. (previously identified as E. sakazakii) are present in bison and bovine feces. In addition to the bison and bovine fecal isolates of Cronobacter spp. found by Nangoh et al., other isolates of Cronobacter spp. were analyzed phenotypically and genetically for biochemical typing and genotyping. The additional isolates include several American Type Culture Collection isolates, an isolate from a neonatal meningitis case, and multiple isolates of various origins received from Cornell University. These isolates were further categorized using four different biochemical tests. The results of these tests placed the isolates into one of the six different species or subspecies within the genus Cronobacter. For genotyping, the isolates were tested for the gene specifically responsible for the a-glucosidase activity. In addition, Pulsed-Field Gel Electrophoresis using two different enzymes, Xbal and Spel, was performed to determine possible genetic similarity of isolates from the bison and bovine feces to other isolates found in food, clinical and environmental settings. The X'baI enzyme showed two Cornell isolates, F6-049 and F6-051, had a high degree of similarity; both of these isolates were from the same clinical source. Isolates from bison and bovine feces, 52 and N72, respectively, have a high degree of dissimilarity to each other, ~ 75%. Isolate 52 showed ~ 35% dissimilarity to an isolate from a food source, and N72 showed ~45% dissimilarity to an isolate from a clinical source. The results using the Spel enzyme showed a wide diversity among the isolates. This study shows that very few of the Cronobacter spp. isolates are closely related and that there is a high level of diversity based on pulse-field gel electrophoresis and biochemical analysis.
Development of Real-Time PCR Method for Detection, Identification and Quantification of Five Different Fusarium Species
(North Dakota State University, 2010) Goswami, Kakolie
Fusarium head blight (FHB) is a fungal disease affecting cereal crops worldwide. FHB involves multiple Fusarium species that create food safety concerns by producing mycotoxins such as trichothecenes, zearalenone and moniliformin. Quantitative methods allowing rapid risk assessment of mycotoxigenic Fusarium species are needed to detect Fusarium spp. Real time quantitative PCR (qPCR) is a fast, sensitive and reliable alternative to conventional culture methods. An objective of the study was to develop TaqMan® based qPCR methods to detect, identify and quantify five different Fusarium species associated with FHB, namely F. graminearum, F culmorum, F avenceum, F poae and F sporotrichioides. The project was initiated with the selection of a protocol for Fusarium DNA extraction. Three different commercially available DNA extraction kits, including FastDNA ®, Qiagen® Blood & Tissue kit and Qiagen® DNeasy Plant kit were evaluated for speed, DNA yield and quality. The results showed that FastDNA ® kit gave the highest DNA yield in least time. TaqMan® Minor Groove Binder (MGB) probes for F culmorum, F poae and F sporotrichioides were developed using qPCR primers from a previous study. For F avenaceum and F. graminearum, TaqMan® based methods already available were used with modified conditions for improved detection. The qPCR methods were tested on a wheat system and the result was a qPCR system that rapidly identified and quantified Fusarium species associated with FHB.
Proteomic and Molecular Analysis of Methicillin Resistance and Selected Toxigenic genes in Coagulase-negative Staphylococcus spp From Food and Animal Sources.
(North Dakota State University, 2010) Aye, Racheal
While most coagulase-negative Staphylococcus (CNS) are apathogenic, recent evidence suggests some food and animal derived CNS isolates may carry and express virulence factors including classical enterotoxins, toxic shock syndrome, and methicillin resistance genes. The present study was designed to assess the potential role of CNS in the epidemiology of foodborne illnesses and to determine the likelihood of food and domestic animals as transmission vehicles of methicillin resistance. Of the animal-derived food samples tested, 27.3% (39/143) were Staphylococcus-positive compared to only 9.5% (23/242) of the plant foods. A total of 92 Staphylococcus spp cultured from 385 food (62/92) and 30 diagnostic animal (30/92) samples were tested by the polymerase chain reaction (PCR) for classical enterotoxin (sea, seb, sec, sec/, and see), toxic shock syndrome toxin-1 (tsst-1), and mecA genes. All PCR-positive isolates were further tested by immunoblotting for production of the corresponding toxin. Susceptibility patterns of both CNS and coagulase-positive Staphylococcus (CPS) were assessed for ~- lactam antimicrobial agents and the isolates analyzed for presence of the mecA gene. Of all study isolates, 20/92 (21.7%) were CPS and 72/92 (78.3%) CNS. Of the 20 CPS isolates, 15.4% (2/13) 5. aureus cultured from steak were sec positive while only 1/7 (14.3%) CPS (5. aureus) from a diagnostic feline sample was positive for both sec and tsst-1 genes. Both toxigenic 5. aureus isolates from steak and a diagnostic feline sample also expressed detectable amounts of SEC and TSST-1 toxins. On the other hand, 1.4% (1/49) of the CNS (5. lugdunensis) from strawberries was positive for the sec gene but negative for SEC toxin and 2/49 (2.8%) of the CNS (5. hominis) gave unexpected base pair PCR products with see primers. All CNS isolates were generally susceptible to test ~-lactam antimicrobial agents; 80% of the CPS isolates were resistant to penicillin and amoxicillin of which 1/20 (5%) were positive for the mecA gene. Based on this data, 3/20 (15%) of CPS and 3/72 (4.2%) of CNS isolates were positive for toxigenic genes thus underscoring the potential role food-derived CNS isolates may have in the epidemiology of foodborne illnesses. Although only 5% of the CPS and none of the study CNS isolates expressed mecA gene, 80% of the CPS were resistant to penicillin, suggesting other mechanisms of drug resistance. The presence of mecApositive CPS and toxin producing Staphylococcus isolates underscore the public health significance of organisms from this genus.
An In-vitro Co-Culture Model to Study the Disruption of the Blood Brain Barrier by Cronobacter sakazakii. (formerly Enterobacter sakazakii)
(North Dakota State University, 2010) Koval, Erin Louise
Cronobacter spp. (formerly Enterobacter sakazakii) is an opportunistic pathogen associated with contaminated powdered infant formula. It causes necrotizing enterocolitis (NEC) and sepsis, which can develop into severe meningitis and brain abscess formation in infants. Very little is known about the specific pathogenic mechanisms of this organism. In this study, we propose a model to investigate the ability of Cronobacter spp. isolates to disrupt the blood-brain barrier. Biofilm formation of the Cronobacter spp. isolates used in the model was also assayed. Secondary mouse endothelial and astrocyte cells were grown to con.fluency on polyethylene terephtalate (PET) membranes (1.0 μm pore size) in 24-well plate hanging culture inserts (Millipore® Billerica, MA) to achieve a culture system similar to the physiological structure of the blood brain barrier in vivo. Initially, a monoculture system was tested containing only endothelial cells and then a co-culture system was developed with both cell types. Selected Cronobacter spp. isolates were added to the cell culture systems. Escherichia coli KI and Kl2 strains were used as positive and negative controls, respectively. Some of the treatments did not have bacteria added to them to serve as cell controls, and membranes without cells were included as media control blanks. The transendothelial electrical resistance (TEER) was measured across the cells to determine if the cell barrier was disrupted. Initially, measurements were taken at 0, 6, 24, and 48 hours after adding bacteria. Due to overall loss of cell integrity at 48 hours, a second experiment was performed where measurements were taken at 0, 6, and 24 hours after adding the bacteria. Biofilm formation was analyzed using a method described by O'Toole (72) and Skyberg (86). The monoculture and co-culture systems worked based on TEER measurements. The positive control (E.coli Kl) and Cronobacter sakazakii isolates disrupted the tight junctions between cells evidenced by significant decrease in TEER over time. The negative control (E.coli K12) did not have any significant effect on the cells. The cell control and negative control (E.coli Kl2) maintained the highest resistance values in both the monoculture and co-culture experiments. The positive control (E. coli Kl) and the Cronobacter sakazakii isolates caused the resistance across the cells to significantly decrease over time in both experiments. According to the results of our biofilm assay, none of Cronobacter spp. isolates used in the culture models formed biofilms. Further testing would need to be done using other biofilm detection procedures to confirm this conclusion Overall, we successfully constructed a co-culture model to depict the BBB. The selected Cronobacter sakazakii decreased resistance in this model of the BBB similar to the positive control Kl E.coli isolate. This assay can be used in future studies to test the potential pathogenicity of Cronobacter spp. bacteria as well as other bacteria involved with central nervous system diseases.
Analysis of Deoxynivalenol and Deoxynivalenol-3-glucoside in Wheat
(North Dakota State University, 2012) Burgess, Kimberly
Deoxynivalenol (DON), a mycotoxin produced in cereal grains infected by Fusarium Head Blight produced by Fusarium graminearium and Deoxynivalenol-3-β-D-glucopyranoside (DON-3G), were studied during processing using LC-MS-MS and GC. DON reduced significantly (P<0.05) 61.8% during milling into flour. Therefore, DON was concentrated mostly in the bran and germ. DON increased 40.8% during the fermentation stage of baking. DON increased in dough more than flour and mixed dough. Milling reduced by 23.7% but fermentation did not. But bread was significantly lower in DON-3G at 0.15 ppm than flour and dough at 0.31 ppm. The baking increased DON and decreased DON-3G showing a difference in stability of the mycotoxins during processing. Enzyme hydrolysis on DON using α-amylase, cellulase, protease, and xylanase, showed a significant increase with cellulase (20.8%), protease (11.4%), and xylanase (35.6%) compared to wheat composite. DON may be bound to the cell wall or protein component of the kernel.
Assessing Microbial Stability and Quality of Green Beans Using Various Home Canning Methods
(North Dakota State University, 2013) Kuchynski, Jenny
Today many consumers follow processing methods recommended either from family members or the internet, which they interpret as being safe. Processing temperature profiles, survival of B. stearothermophilus spores, texture, and color of green beans processed under four home canning methods were assessed. The products were processed using pressure, boiling water bath, steam, or oven canning methods. Pressure canning produced the greatest microbial reductions but this method resulted in the lowest bean quality. The boiling water bath, steam, and oven canning were found to be less safe because the product temperature never achieved 100°C and the resulting microbial counts, >1.7 log CFU/ml, were observed after processing. However, green bean quality was better than pressure canning, with beans from steam canning having the firmest texture and best green color. Although better green bean quality results were observed from internet or family based methods, their safety is questionable considering the high microbial survival.
The Occurrence of Shiga-Toxin Producing Escherichia Coli (Stec) and Salmonella Species in Cattle Feedlot Runoff
(North Dakota State University, 2013) Tabe, Nessie Nanyongo
Zoonotic foodborne pathogens such as shiga-toxin producing Escherichia coli (STEC) and Salmonella on farm environments can potentially contaminate organic manure or agricultural watersheds and subsequently fresh produce during fertilization or irrigation. This study investigated the occurrence of STEC and Salmonella serotypes in cattle feedlot runoff samples in two feedlots in North Dakota. Using standard laboratory culture methods this study reported a 39% prevalence of STEC O45, 33 % (O103), 31 % (O157), 27 % (O121), 16 % (O26), 10% (O111), 10% (O113), 10 % (O145) and 39.7 % Salmonella. Additionally, occurrence of some STEC serotypes was influenced by feedlot (O111 and O121), sampling location in relation to vegetative filter strips (O157), and sampling time (O45 and O121). Although this study was the first to report occurrence of STEC serotypes including non-O157 serotypes in cattle feedlot runoff, further studies are needed to quantify the pathogen load in runoff prior to disposal.
The Use of Communication Strategies to Influence Stakeholders to Implement Food Safety Management Systems in Small Custom-Exempt Meal Plants
(North Dakota State University, 2014) Rathnasinghe, Shalindra Suresh
This exploratory study used interviews to understand the culture and communication patterns of the stakeholders, employers, and employees. Interviews revealed that the topic of Food Safety was a very sensitive one as many were reluctant to share information. The study found that direct informal communication strategies are the best method to communicate custom-exempt meat plants. These communication strategies can be used to influence food safety practices.
Fate of Deoxynivalenol and Deoxynivalenol-3-Glucoside during the Malting Process
(North Dakota State University, 2015) Jiang, Wei
Deoxynivalenol (DON) is commonly found on small grains and causes food safety issues. Deoxynivalenol-3-Glucoside (DON-3-G) is a conjugate, formed as a defense response by the host plant. Past studies have shown both to be present in Fusarium infected small grains, and processed products like beer, but there is limited information on DON-3-G in malt. Objectives were to determine the levels of DON-3-G in barley and wheat, and to study its fate during malting of inoculated and commercial samples. Commercial barley and wheat samples were used to determine levels in naturally infected grain. During malting, barley DON declined 48% on average, but DON-3-G increased by 115%. Both compounds increased in malted wheat. The genotype x crop year interactions were significant for both toxins, indicating that the genotypes did not respond similarly in the two years. The potential for large amounts of DON-3-G to be formed during malting has not been reported.
Wheat Dockage Content: Analysis of Dockage and Its Relation to Fusarium Head Blight
(North Dakota State University, 2016) Alhumaid, Taghrid Saleh
Hard red spring wheat crop grown in different locations in the US were surveyed for the mycotoxin deoxynivalenol (DON). DON is often found in wheat that is infected with the plant fungal disease Fusarium head blight. The contamination of wheat by DON is a major wheat industry concern since it affects human and livestock health. Furthermore, DON reduces wheat grain yield and quality. In this study, DON was measured using gas chromatography with electron capture detection (GC-ECD) in 1353 HRS wheat samples collected from 2013-2015. Results indicate that there was positive significant correlation (P<0.001) between DON content and damaged kernels (0.635) and total defects (0.445). However, for the three-year average, DON content had a weak positive significant correlation (P<0.001) with the percentage of wheat dockage (0.111). Overall, DON production had an effect on kernel damage and total defects, but DON production was not impacted by the percentage of wheat dockage.
Growth of Fusarium graminearum and Production of Trichothecenes During the Malting of Winter Rye and Triticale
(North Dakota State University, 2019) Tang, Ruoling
There is growing interest in malting and brewing with rye. However, previous research has shown a propensity for the development of deoxynivalenol (DON) in rye malts, even when levels on the grain is low. The main objective of this study was to assess the growth of F. graminearum and development of trichothecenes during malting of rye. Infected samples were obtained from 2016 variety trails in Minnesota. While DON levels were generally below 0.2 mg/kg, an average increase of 41 % was seen after malting. The most significant increases in DON were at three days of germination. Fusarium Tri5 DNA levels were observed to increase at two days. When single kernels were tested, most were free from DON. Levels in the bulk grain sample were due to a small number of highly contaminated kernels. In the malted samples, a greater portion of kernels contained DON, and overall levels were much higher.
Compensation for the Effectiveness of Risk Mitigation Strategies for Fusarium Head Blight(FHB) and Deoxynivalenol(DON)
(North Dakota State University, 2019) Lyonga, Nathalie Ndedi
Food safety related problems are one of the biggest challenges worldwide. DON is produced by Fusarium species which causes the well-known Fusarium Head Blight (FHB) of wheat and barley. FHB outbreaks have led to variability in yield and revenue losses over the years. The main objective of my thesis was to quantify risk premiums at the farm level and with industry impact, to determine the effectiveness of FHB/DON mitigation strategies over time from 1997 to 2014. Data on revenue losses ($million) were obtained from USDA-ERS and was simulated using a risk analysis software called @RISK 7.5. The sample data was simulated 10,000 times to obtain a population. Risk premiums were calculated for each year and for each crop over time and graphs were plotted. Trends in risk premiums showed an overall decrease from 1997 to 2014, indicating that variability of losses have reduced and that the management practices have been effective.
Strategies to Inhibit the Formation of 3-Monochloropropane Diol During Deep-Fat Frying
(North Dakota State University, 2020) Ye, Qionghuan
3-monochloropropane-1,2-diol or 3-chloropropane-1,2-diol (3-MCPD) and glycidol are the most commonly occurring group of thermal process contaminants which are considered as “possible human carcinogen” and “probably carcinogenic to humans”, respectively. Potato strips prepared from three different potatoes cultivars (Russet Burbank, Ranger Russet, and Umatilla Russet) grown in North Dakota from the crop year 2018 were fried with vegetable oil at 190 ºC, respectively, for five consecutive days (8 h/day). The dynamic changes of 3-MCPD and glycidol equivalents were investigated during deep-fat frying. 3-MCPD equivalent in oil and potato strips decreased with increased frying time. Meanwhile, the content of glycidol equivalent increased with increased frying time. The major 3-MCPD and glycidol equivalents that were detected in the fried potato strips were those that migrated from the oils during frying. The application of absorbents, i.e., Magnesol and Celite, achieved the mitigation of 3-MCPD and glycidol in frying oil.
Quantitative Analysis for Measuring Lower Levels of Fusarium Mycotoxins in Wheat and Barley Using High-Resolution Mass Spectrometry
(North Dakota State University, 2020) Sarkate, Pradeep Samadhan
This project involved the application of Quadrupole Time of Flight (QTOF) technology in quantitating the low concentrations of multiple Fusarium mycotoxins in barley and wheat, also focused on simplified sample extraction protocols such as ‘dilute and shoot.’ Ground samples of wheat and barley were extracted with acetonitrile-water-acetic acid solution (70:29:1 v/v/v). The quantitation was performed using a post spiking matrix-matched calibration curve approach. The method was linear over the range of 1.56 – 100 μg/kg for the toxins deoxynivalenol (DON), deoxynivalenol-3-glucoside (D3G), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), diacetoxyscirpenol (DAS), fusarenon-X (FUS-X), nivalenol (NIV), Neosolaniol (NEO), T2, and HT2 toxin. Zearalenone (ZEA). The recovery of the 11 mycotoxins in wheat and barley matrices at two levels were within 60 - 130.1%, and the relative standard deviation (RSD) of the replicate sample assay fell within 5 to 40%. Overall, this method was successfully validated for all the Fusarium toxins.

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