The consumption of seafood in the United States has increased rapidly in recent years due to high quality protein and health benefits of seafood. Seafood can be a carrier for bacteria normally distributed in the marine environment and, in some cases, can be contaminated by human pathogens. Therefore, there is a potential health risk if seafood is consumed raw or undercooked. However, information regarding prevalence of foodborne pathogens in retail seafood products and the ability of pathogens to survive in the products during refrigerated and frozen storage is limited. The objective of this study was to generate such information for a better understanding of distribution of foodborne pathogens in seafood products and provide data which might be used for risk assessment of foodborne infection associated with seafood consumption. A total of 45 seafood products were collected from local retail stores and analyzed for aerobic plate counts (APC) and psychrotrophic bacterial counts (PBC) as well as presence of foodborne pathogens, including Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus according to procedures described in the U.S. Food and Drug and Administration Bacteriological Analytical Manual (BAM). Presumptive isolates for each foodborne pathogen were further characterized by biochemical reactions using commercial identification kits and confirmed with polymerase chain reaction (PCR) assay. The samples had bacterial populations ranging from 1.90 to 6.11 CFU/g for APC and from 2.00 to 6.78 CFU/g for PBC. According to the microbiological criteria of International Commission on Microbiological Specifications for Foods (ICMSF), all 45 samples were considered acceptable quality (APC < 10⁷ CFU/g, E. coli < 3 MPN/g) with most samples (93.3%) being good quality (APC < 5 × 10⁵ CFU/g, E. coli < 3 MPN/g). No E. coli O157:H7, Salmonella, S. aureus, V. parahaemolyticus, and V. vulnificus was detected in any samples. Two previously frozen shrimp products (4.4%) were confirmed to carry L. monocytogenes. Studies of growth and survival of L. monocytogenes (3 strains), S. aureus (2 strains), and Salmonella (2 serovars) in raw yellowfin tuna meat stored at 5 - 7 °C for 14 days revealed that L. monocytogenes had the ability to multiply in the tuna meat during refrigerated storage while populations of S. aureus and Salmonella were reduced by 1 to 2 log CFU/g after 14 days at 5 - 7 °C. Studies of holding raw yellowfin tuna meat contaminated with L. monocytogenes, S. aureus, and Salmonella at -18 ± 2 °C for 12 weeks observed that all three pathogens, except Salmonella Newport, in tuna samples survived the frozen storage with less than 2- log of reductions in the populations over 12 weeks of storage. No viable cell of Salmonella Newport was detected in samples after 42 days storage at -18 °C. Raw seafood can be a carrier of foodborne pathogens, particularly L. monocytogenes, and many foodborne pathogens can survive in frozen products for several months. Consumption of raw or undercooked seafood products may lead to human infection if the products are contaminated with pathogens. Therefore, sanitation standard operating procedure (SSOP), good manufacturing practice (GMP) and hazards analysis and critical control points (HACCPs) programs shall all be implemented in the seafood industry to prevent seafood products from being contaminated with foodborne pathogens during handling and processing. Moreover, proper storage of raw seafood products and avoiding cross-contamination during handling at the retail levels also helps to minimize risk of human infection associated with ready-to-eat products. / Graduation date: 2013

Foodborne diseases form a large group of infectious diseases. These substantial diseases are occurring more in developing countries but they do not avoid developed countries. The originators of the disease are bacteria and their toxins, viruses, parasites and prions, whose source is an infected person or animal. The disease is passed on by contaminated soil, contaminated water and food, sometimes contaminated items. Greatest importance in the prevention of foodborne disease are nonspecific measures, protection of drinking water, safe food production, waste disposal, functional sewer system, rodenticides and insecticides measures and of course the principles of personal hygiene, certain standards of hygiene and health education of the population. Intestinal infections are preventable. The consumer can effectively protect by following the fundamental principles established by the World Health Organization. The aim of this study was to explore knowledge of foodborne disease prevention measures and compliance with these principles by adolescents in the Milevsko region. The theoretical part of the study summarizes the basic information about intestinal diseases and describes their effect on health from a variety of perspectives. The preventive measures which everyone should be aware of and apply them in everyday life to protect health are described in great detail. The paper summarizes the information about the process of spreading foodborne diseases and epidemiological measures focused on supply, routes of transmission and susceptible individuals. The theoretical part includes basic information about specific intestinal infections which are most significant for the population in the Czech Republic. Briefly described are treatment of these diseases and the most important legislative measures. In the research part quantitative research was applied by questionnaire. The research groups were students of higher secondary vocational schools and vocational schools in the region Milevsko. Three hypotheses were formulated. Statistical hypothesis testing was performed using the t test. The values of the achieved significance level are compared with a predetermined level of significance. The first hypothesis assumed that knowledge about prevention of intestinal diseases is higher among girls than boys. This statement has not been confirmed. Knowledge of the principles of prevention does not differ according to the criteria of gender. Two further hypotheses assumed that the observance of hygienic measures in the prevention of foodborne disease is higher among girls than among boys and higher for high-school graduates than apprentices. These hypotheses are valid. Precautionary principles are indeed respected more by girls than boys and high-school graduates than apprentices. The carried out research shows that adolescents in the region Milevsko have a good knowledge of foodborne infections and do known also essential preventive measures. However, knowledge of these rules does not mean they are applied in practice. The results showed that some rules, such as washing fruits and vegetables before eating, adequate boiling or roasting of food, especially hand hygiene before eating food or after contact with an animal are observed by a smaller percentage of respondents than that reported knowledge of these principles. A positive finding is that respondents comply with certain rules, without being aware of them, such as the principle of proper preservation of precooked food. Foodborne diseases are diseases which can be prevented in compliance with precautionary principles. Especially in the Czech Republic, where nonspecific principles are at a high level. It is up to each user/consumer how consistent these principles will be. Respecting preventive measures is a prerequisite for reducing the incidence of foodborne disease in the population.

Doctor of Philosophy / Department of Food Science / Randall K. Phebus / Intervention techniques to control foodborne pathogens, and rapid identification of pathogens in food are of vital importance to ensure food safety. Therefore, the first objective of this research was to study the efficacy of radio frequency dielectric heating (RFDH) against C. sakazakii and Salmonella spp. in nonfat dry milk (NDM) at 75, 80, 85, or 90°C. Using thermal-death-time (TDT) disks, D-values of C. sakazakii in high heat (HH)- and low heat (LH)-NDM were 24.86 and 23.0 min at 75°C, 13.75 and 7.52 min at 80°C, 8.0 and 6.03 min at 85°C, and 5.57 and 5.37 min at 90°C, respectively. D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75°C, 10.45 and 12.54 min at 80°C, 8.63 and 8.68 min at 85°C, and 5.82 and 4.55 min at 90°C, respectively. The predicted (TDT) and observed (RFDH) destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the organisms' behavior was similar regardless of the heating system (conventional vs. RFDH). However, RFDH can be used as a faster and more uniform heating method for NDM to achieve the target temperatures. The second objective of this research was to study if hyperspectral imaging can be used for the rapid identification and differentiation of various foodborne pathogens. Four strains of C. sakazakii, 5 strains of Salmonella spp., 8 strains of E. coli, and 1 strain each of L. monocytogenes and S. aureus were used in the study. Principal component analysis and kNN (k-nearest neighbor) were used to develop classification models, which were then validated using a cross-validation technique. Classification accuracy of various strains within genera including C. sakazakii, Salmonella spp. and E. coli, respectively was 100%; except within C. sakazakii, strain BAA-894, and within E. coli, strains O26, O45 and O121 had 66.67% accuracy. When all strains were studied together (irrespective of their genera) for the classification, only C. sakazakii P1, E. coli O104, O111 and O145, S. Montevideo, and L. monocytogenes had 100% classification accuracy; whereas, E. coli O45 and S. Tennessee were not classified (classification accuracy of 0%).

Master of Science / Food Science Institute,Human Nutrition / Valentina M. Remig / Foodborne illness is a serious health problem in the United States, and especially in the mature adult population. This research examined food safety knowledge of mature Kansans(aged 55 years and older) in three important constructs (handwashing, food handling, and food preparation), their knowledge of foodborne illness symptoms, and their food safety concerns while eating away from home. One-hundred and forty participants completed a self-administered questionnaire containing ten food safety knowledge questions representing sixty answer options. Replies to those questions were compared by age, gender, geographic location,and educational attainment. Results indicated that geographic location was statistically significantly related to food safety knowledge; however, age, gender, and education had little to moderate association. Additional findings revealed food safety knowledge was not associated with participants’ level of food safety concern while eating away from home. In conclusion, the findings revealed that mature Kansans possessed general food safety knowledge; however, some responses indicated mature adults did not fully understand certain food safety protocols. Areas identified as needing further attention included appropriate hand drying and surface cleaning, safe food and refrigerator temperatures, proper thawing practices, as well as safe leftover and melon preparation.

Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.

Spores of foodborne pathogens such as Clostridium botulinum, Clostridium perfringens and Bacillus cereus are widely distributed in nature. Presence of those spores in food products, particularly C. botulinum spores in vacuum packed, ready-to-eat low-acid products, is a great safety concern. The research here described is a first effort towards understanding the role of the spore coat proteins in the inactivation of bacterial spore using high pressure processing. This study proposes a coat protein solubilization methodology using non-ionic detergents minimizing protein damage and compatible with spectroscopy methods. The methodology developed here was compared with approaches proposed in the literature with respect to protein yield, protein fractions identified, amino acid composition and suitability with spectroscopy techniques for the further analysis of coat proteins. Bacillus subtilis ATCC 6633 spore coat proteins were solubilized (n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance (ANOVA) showed no significant (95% confidence) differences between the three repetitions of the three spore coat protein solubilization methods. Protein yield was significantly larger (95% confidence) when using UDS at 70C as compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein solutions consisted of five major and six minor proteins ranging 6 to 65 kD while the OGP solution appear to consist of four major and nine minor bands in the same mw range. Amino acid analysis of the protein extracted by the OGP method was conducted using reverse phase HPLC (RP-HPLC) and compared with published information. The OGP spore coat protein solution showed a higher proportion of aspartate, glutamate, alanine and tyrosine. Pressure, heat and time effects were studied on spore coat proteins obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores. Pressure treatments of spores, and of extracted spore coat protein solutions, at 50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C along with appropriate heat- and pressure-only controls and untreated sample, were used to study the effect of pressure, heat and time on spore coat proteins. Both spore coat protein solubilization procedures showed a significant reduction in protein yield for pressure-only, heat-only and pressure/heat treated spores when compared with untreated spores. When OGP-solubilized proteins from untreated spores were pressure treated, SDS-PAGE profile showed an increasing overall band intensity with increasing pressure and time. In the case of protein solution obtained from pressure-treated spores the electrophoretic pattern showed the loss of higher molecular weight proteins. The significance of this study is that for the first time we have observed extensive changes on spore coat proteins caused by pressure, as well as heat treatments. Future studies will examine what is the probable physiological role of the proteins damaged by these physical treatments. An advantage of the protein solubilization here developed will allow the application of spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003

Water quality, and therefore human health, may be significantly affected by the presence of pathogenic enteric microorganisms derived for improper disposal of wastewater to aquatic environments. Detection of waterborne viruses is complex due to the difficulties in concentrating the sample and then in detecting the virus by cell culture or molecular techniques. Methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years. The U.S. Environmental Protection Agency requires the use of 1MDS electropositive filters for concentrating enteric viruses from water; however, these filters are expensive for routine viral monitoring. The NanoCeram® filter, an electropositive cartridge filter, has been proposed as a new alternative for large volumes of water. The objective of the study was: to evaluate the effectiveness of NanoCeram® filters for the concentration of poliovirus-1 from wastewater samples and compare to 1MDS cartridge filters. This study suggested that NanoCeram® filters are a viable alternative to the use of 1MDS filters for viral monitoring in surface waters and wastewaters. L. monocytogenes outbreaks with Latin-style soft cheese have been well-documented; however, more information to characterize the human health risk associated with the consumption of queso fresco (QF) using unpasteurized milk is needed. The objectives of the study were: i) to evaluate the prevalence of Listeria, Escherichia coli, Salmonella and fecal coliforms in QF obtained from markets in the northwestern state of Sinaloa, Mexico, and ii) to address the human health impact associated with the consumption of QF contaminated with L. monocytogenes using quantitative microbial risk assessment (QMRA). The study suggested that QF produced in Culiacan, Sinaloa, Mexico have microbial loads above the maximum values recommended by the Official Mexican Regulations; and QMRA can be used to interpret microbial contamination data for impacts on public health.

Foodborne pathogens are a threat to public health worldwide. Because many consumers prefer natural compounds to synthetic additives, research on safe plant-derived compounds with antimicrobial activity against foodborne pathogens is vital. The aim of this investigation was to evaluate the antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, trans-cinnamaldehyde, citral) and plant-extracts such as green tea, apple skin extract, black and decaffeinated black tea, grapes seed and pomace extracts against foodborne bacteria. Salmonella enterica serotype Typhimurium DT104, and serotype Newport, were selected conducting an antibiotic screening on 23 Salmonella isolates using seven antibiotics to determine antibiotic resistance. Listeria monocytogenes (strain 101M; beef and pork sausage isolate; resistant to antimicrobials in past investigations) was included to represent gram-positive bacteria. Escherichia coli O157:H7 virulent isolates (932- apple juice isolate; ATCC 35150- human isolate; F4637- sprouts isolate; used as a cocktail) were selected after conducting a Multiplex PCR over nine E. coli O157:H7 isolates to detect shiga-toxin 1 and 2 genes. All antimicrobials were evaluated in vitro in phosphate buffered saline. In general, all pathogens were more susceptible to essential oils and their active components, than powder extracts. The most active antimicrobials from each category were directly applied on foods. The activity of oregano oil (0.5%) and green tea (3%) was evaluated against S. Typhimurium on chicken and S. Newport on tomatoes and sprouts, and the results showed that oregano oil was more effective. In addition, baby spinach leaf samples inoculated with green fluorescent protein labeled S. Newport were examined under confocal scanning laser microscope before and after antimicrobial treatments. Antimicrobial experiments against L. monocytogenes on sprouts, ham and bologna, carvacrol at 0.5% and grape seed extract at 3% were used and carvacrol showed better activity. Antimicrobial activity against E. coli O157:H7 was tested on romaine lettuce, spinach and ground beef using oregano oil at 0.5% and green tea at 3%. Both compounds were effective showing no recovery of E. coli O157:H7 from lettuce and spinach; however, was not reduced in ground beef. Antimicrobial plant compounds have the potential for reducing foodborne pathogenic bacteria on/in various foods.

Arcobacter spp., anteriormente conhecido como Campylobacter aerotolerante, é considerado um gênero bacteriano que inclui espécies consideradas patógenos emergentes que podem ser veiculados por alimentos. O gênero Arcobacter tem sido associado a gastroenterites, diarreia persistente e bacteremia em humanos. É uma bactéria Gram negativa, termosensível, embora possa sobreviver à 4°C. No Brasil, há poucos estudos de ocorrência de Arcobacter em alimentos, inclusive os de origem animal, especialmente os mais consumidos, como as carnes de frango e suína. Existem estudos pontuais de sua ocorrência em produtos resfriados, como cortes e em carcaças de frango resfriadas do varejo. O objetivo deste estudo foi avaliar a ocorrência de Arcobacter spp. em cortes e carcaças de frango refrigeradas e congeladas do varejo e em coxas de frango livres de antibiótico e orgânico refrigeradas provenientes de abatedouro, por técnica de isolamento convencional com posterior confirmação do gênero por reação de polimerase em cadeia (PCR). Foram analisadas 153 amostras de carne de frango, das quais 39,21% (59/153) resultaram positivas para o gênero Arcobacter. Foi obtido o total de sessenta e quatro isolados positivos para Arcobacter spp., que corresponderam a 89,06% (57/64) de A. lacus, 4,7% (3/64) de A. thereius, 3,12% (2/64) de A. butzleri, e 3,12% (2/64) de Arcobacter spp. espécie não identficada até o momento. Foram realizadas análises fenotípicas de resistência a 12 antibióticos com 34 isolados, previamente selecionados, de quatro diferentes fontes de carnes de frango obtidos nesse trabalho, e de três linhagens utilizadas como controle positivo de Arcobacter. A resistência fenotípica frente aos antimicrobianos foi de 100% para ácido nalidíxico e clindamicina, 29,73% para eritromicina, 24,32% para canamicina, 21,62% para tetraciclina, 18,42% para cloranfenicol, 13,51% para gentamicina, 8,11% para estreptomicina, 5,41% para azitromicina e ciprofloxacina, 2,10% para vancomicina e 0,00% para ampicilina. / Arcobacter spp., previously known as aerotolerant Campylobacter, is considered a bacterial genus that includes species considered emerging pathogens that can be transmitted by food. Arcobacter has been associated with gastroenteritis, persistent diarrhea and bacteremia in humans. It is a gram negative, thermosensitive bacterium, although it can survive at 4 ° C. In Brazil, there are few studies of the occurrence of Arcobacter in animal products including those of animal origin, especially those most consumed, such as poultry and pork. There are occasional studies of their occurrence in cooled products, such as cuts and in refrigerated chicken carcasses. The objective of this study was to evaluate the occurrence of Arcobacter spp. in refrigerated chicken cuts and carcasses of the retail and in chicken thighs free of antibiotic and organic refrigerated from slaughterhouse by conventional isolation and genotyping by polymerase chain reaction (PCR). A total of 153 chicken meat samples were analyzed, of which 39.21% (59/153) were positive for the Arcobacter genus. A total of sixty-four isolates positive for Arcobacter spp., corresponding 89.06% (57/64) of A. lacus, 4.7% (3/64) of A. thereius, 3.12% (2/64) of A. butzleri, and 3.12% (2/64) of Arcobacter spp. species not yet identified. Phenotypic resistance analyzes were performed on 12 antibiotics with 34 isolates, previously selected from four different sources of chicken meat obtained in this study, and three strains used as positive control of Arcobacter spp. Phenotypic resistance to antimicrobials were 100% for nalidixic acid and clindamycin , 29.73% for erythromycin, 24.32% for kanamycin, 21.62% for tetracycline, 18.42% for chloramphenicol, 13.51% for gentamycin, 8.11% for streptomycin, 5.41% for azithromycin and ciprofloxacin, 2.10% for vancomycin and 0.00% for ampicillin.

Em um total de 90 amostras de hortaliças, incluindo 30 de alface, 30 de agrião e 30 de escarola, foi verificada a ocorrência de Aeromonas do grupo A. hydrophila, empregando-se os métodos de semeadura direta em ágar amido-amplicilina (contagem) e após enriquecimento em caldo tripticase-soja adicionado de ampicilina (teste de presença/ausência). As incubações foram feitas a 28&#186;C, durante 24 horas. A presença dessas bactérias foi detectada em 43 (47,8%) das amostras analisadas, com contagens variando de < 102 a 2, 0x 106UFC/g. As amostras de agrião foram as que revelaram, na contagem, com maiores números de Aeromonas spp. Das 43 amostras positivas para Aeromonas spp. 9 (21,0%) revelaram-se com números superiores a 104 UFC/g. sendo que 7 eram de agrião . Dentre as amostras de hortaliças analisadas, as de agrião revelaram-se com positividade para Aeromonas do grupo A. hydrophila (70,0%) significativamente maior em relação às de alface (43,3%) e de escarola (30,0%) a nível de 5%. Não foram observadas diferenças significativas entre as positividades obtidas através do método de semeadura direta em placas e do teste de presença/ausência para as amostras de alface e de agrião. Para as amostras de escarola, a positividade foi significativamente mais alta no teste de presença/ausência. Do total de 143 cepas confirmadas como sendo do gênero Aeromonas, 138 (96,5%) eram de A. caviae, 4 (2,8%) de A. hydophila e 1 (0,7%) que, pelas suas características, foi considerada como Aeromonas atipica. Dos resultados obtidos, pode-se depreender que as hortaliças dos tipos analisados, alface, agrião e escarola, dado os níveis de contaminação observados, podem representar risco aos consumidores. / A total of 90 retail vegetable samples, including 30 of lettuce, 30 of water-cress and 30 of escarole were examined for the presence of Aeromonas of the A. hydrophila group, using two different isolation methods. One of the methods envolved direct plating on starch-ampicinin agar for the purpose of enumeration and the other one, after enrichment in trypticase-soy broth with ampicillin, for detection, both using 24 hour incubation at 28&#176C. Aeromonas spp. Were detected in 43 (47.8%) samples and their numbers varied from less than 102 up to 2.0x106 CFU/g. The water-cress samples were the ones to show greater numbers of Aeromonas spp. The counts of 9 (21%) of the 43 positive samples exceeded 104 CFU/g, 7 of them consisting of water-cress. The number of water-cress positive samples (70.0%) was significantly higher at 5% than those of lettuce (43.3%) and those of escarole (30.0%). No significant differences were found in relation to positivity for Aeromonas spp. Between both isolation methods used, regarding the lettuce and the water cress samples. On the other hand, with respect to the escarole samples, positivity was significantly superior for the isolation method envolving enrichment. In therms of species level identification, among 143 strains confirmed as being Aeromonas spp., 138 (96.51%) were A, cavia, 4 (2.81%) were A, hvdrophila and 1 (O,7%) was considered as atypical due to its different biochemical profile. The results show that the vegetables examined may represent risk to consumers in terms of presence and numbers of Aeromonas of the A, hvdrophila group.