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Various control methods used in the meat and poultry processing environment to mitigate listeriosis were evaluated using a dynamic in-plant Monte Carlo model. These control methods included food contact surface testing, sanitation, post-processing lethality treatment, and product formulation with microbial growth inhibitors. The dynamic in-plant model served as an input into the risk assessment model developed by the FDA and FSIS in 2003 which predicts the number of deaths and illnesses resulting from the use of each control method. The use of growth inhibitors combined with a post-processing lethality step was estimated to save over 200 more lives than the FSIS proposed minimum sampling standard. An analysis of data collected by the National Alliance for Food Safety and Security (NAFSS) found that retail-sliced deli meats have a greater prevalence and concentration of L. monocytogenes than prepackaged deli meats. Cross contamination at the retail level is suspected due to clustering of sample positives by store and the influence of sampling time of day on the prevalence of L. monocytogenes. The comparative risk of Listeria monocytogenes in retail sliced versus prepackaged deli meats was evaluated using a modified version of the 2003 FDA-FSIS risk assessment model which considered slicing location and the use of growth inhibitors. The comparative risk ratio for the number of deaths from retail-sliced versus prepackaged deli meats was found to be 9.1 and retail-sliced product with a growth inhibitor was found to be at greater risk for listeriosis than prepackaged product without growth inhibitor. / Master of Science

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.

Listeria monocytogenes is a foodbome pathogen that can cause severe illness and even death. It is found in dairy and meat products. The focus is on rapid detection since conventional methods are time consuming (4-5 days). Pre-enrichment steps, as part of those methods, are time consuming. Our objective was to develop a detection system without a pre-enrichment step, giving a final result within 2 to 4 h. In the concept of "the need for speed," a detection system with an antibody-based capture technique, followed by polymerase chain reaction (PCR), was developed. Glass beads coated with a Listeria polyclonal antibody were added to the food sample. After a static incubation/capturing step, beads-cell complexes were separated from the food, and boiled to lyse the cells and release the DNA. In a final PCR/electrophoresis step the DNA samples were analyzed. The use of a flow-based capturing system (ImmunoFlow) was also investigated. Using a bead-antibody complex in this ImmunoFlow setup has several advantages, including the possibility of concentrating the microorganisms out of large food samples (with flow through setup), the exclusion of a pre-enrichment step, and the potential for automation. Besides buffer solution (Tris), different kinds of milk, e.g., pasteurized, Ultra High Temperature (UHT), and raw milk, were also investigated. The detection limit in buffer solution was 1 x 106 CFU/ml no matter if the ImmunoFlow system or the static incubation was used. For the different pasteurized milk samples, the detection limit varied between 1 x 107 and 1 x 108 cells/ml in the static procedure. For UHT and raw milk, however, capturing of Listeria monocytogenes cells was not possible in the static or the ImmunoFlow setup. In conclusion, we developed a rapid and specific detection system for Listeria monocytogenes at high concentration in pasteurized milk using a static capturing procedure. The total test time for this detection system is less than 4 h, which is much faster than the present detection systems (which are using an enrichment step prior to testing). Implementing a real-time PCR system after capture would further reduce this detection time.

Les toxi-infections dorigine alimentaire ont un impact important sur la santé humaine. Cette étude cible les principaux agents bactériens pathogènes pour lhomme transmis par les denrées alimentaires dorigine animale en Europe et aux USA, à savoir Salmonella et Campylobacter, ainsi que leurs éventuels germes index. Globalement, trois types de surveillance permettent de vérifier lefficacité des mesures prises dans le but de diminuer les zoonoses. La première est le suivi de lhygiène et dindicateurs par le dénombrement des flores indicatrices, surtout de contamination fécale. La deuxième est la surveillance dindex pour certains pathogènes tels que Salmonella et Campylobacter. La troisième est la recherche ou le dénombrement direct des flores pathogènes. Le type de surveillance est déterminé par les objectifs visés. Lobjectif principal de ce travail était de déterminer la pertinence de lutilisation dindicateurs de contamination fécale pour surveiller et maîtriser la contamination des filières belges de production de produits carnés par Salmonella et Campylobacter. Dans un premier temps, un plan de surveillance des filières de production et de transformation des viandes a dû être mis en place et optimisé. Les différents paramètres à préciser étaient le choix des agents pathogènes devant faire lobjet dun monitoring dans chacune des filières, et le choix de la méthodologie déchantillonnage et danalyse. Cette étude a montré la qualité et la représentativité des plans de surveillance mis en place en Belgique pour la production carnée. Ils permettent en effet de nombreux types dinterprétation des résultats et cadrent parfaitement avec les programmes de contrôle intégrés pluriannuels imposés récemment par la Commission européenne. Dans un deuxième temps, une étude spécifique a évalué la pertinence de la méthode belge de prélèvement des carcasses de porc (par écouvillonnage) par rapport à la méthode de référence européenne (destructive), qui a fait lobjet dun nouveau règlement européen fin 2005. Une comparaison entre les deux méthodes pour le dénombrement dE. coli et de germes aérobies totaux ainsi que la recherche de Salmonella et de Campylobacter a montré lefficacité de la méthode belge de prélèvement sur les carcasses de porc. Dans un troisième temps, le plan de surveillance mis en place a permis de suivre lévolution de la contamination par Salmonella et Campylobacter des viandes de bovin, de porc et de volaille depuis 2000. Cette étude a confirmé la forte contamination par Salmonella et Campylobacter des carcasses et viandes de volaille. Elle a également montré la plus faible contamination par Campylobacter et, dans une moindre mesure, par Salmonella, des échantillons issus de bovins et de porcs. Une diminution significative de la contamination par Salmonella de la viande de porc et de la prévalence de Campylobacter sur certains échantillons de volaille a été observée entre 2000 et 2003. Cette étude a également souligné limportance de disposer de données nationales de contamination par les microorganismes pathogènes des aliments. Elles permettent en effet de suivre lévolution et de la comparer dun point de vue international. Ces données pertinentes peuvent être utilisées pour une évaluation quantitative des risques et pour la détermination de critères microbiologiques adaptés. Dans un quatrième temps, cette étude a évalué la pertinence du suivi des principaux indicateurs dhygiène et des bonnes pratiques lors des processus de transformation (les germes aérobies totaux, les entérobactéries et les E. coli ), ainsi que de leur qualité dindex des principaux agents pathogènes non sporulés dorigine digestive. Ce chapitre propose une procédure pour fixer des critères dhygiène des procédés adaptés à la Belgique pour les carcasses et la viande de buf, de porc et de volaille. Cette étude a montré que la situation belge en matière de microorganismes indicateurs est comparable à la situation décrite par plusieurs autres études publiées, et que la méthode de détermination de critères dhygiène des procédés en se basant sur les résultats des plans de surveillance est à privilégier pour aider le secteur de production à améliorer la qualité microbiologique de ses produits. Cette approche peut aider à la diminution de la contamination par les agents pathogènes dorigine digestive mais ne peut, à elle seule, donner des garanties de maîtrise de ces dangers. Enfin, il peut être conclu que le dénombrement dE. coli est très utile pour la détermination de lhygiène des procédés de production de viande en tant quindicateur de contamination fécale et en tant quindex dagents zoonotiques dorigine intestinale tels que Salmonella et Campylobacter. Cependant, la recherche, voire le dénombrement direct des agents pathogènes reste nécessaire pour évaluer le risque et sassurer de lefficacité des mesures de maîtrise. Lensemble des résultats de cette étude et les stratégies appliquées sont également très utiles aux autorités belges dans le cadre de négociations au niveau européen pour la détermination de critères microbiologiques. Il est donc important que des plans de surveillance adaptés aux exigences réglementaires européennes continuent de suivre lévolution des agents pathogènes en Belgique en respectant les objectifs fixés au niveau européen. De plus, les critères dhygiène des procédés doivent être régulièrement revus, sur base des résultats des plans de surveillance. Il faut également tenir compte des indicateurs les plus pertinents pour surveiller et maîtriser la contamination par des microorganismes pathogènes émergents, ou dautres agents pathogènes. Ces critères dhygiène des procédés devraient être repris dans les guides dautocontrôle sectoriels. La méthodologie de détermination de critères de sécurité des aliments en se basant sur une évaluation quantitative du risque, conformément aux directives du Codex Alimentarius, devrait être développée, standardisée et appliquée aux niveaux européen et belge. Dans ce cadre, disposer de plans de surveillance performants et quantitatifs sera essentiel pour alimenter les modèles à léchelle de la Belgique.

Raw and processed foods are vulnerable to contamination during their production, distribution and sale. Thus, a wide variety of chemical preservatives are used in the food industry to prevent the growth of food spoilage and pathogenic bacteria. However, health and economic concerns have led to an intensive search for natural alternatives, such as plant extracts, that can safely be used as substitutes for synthetic antimicrobials and preservatives to partially or completely inhibit the growth of bacteria. This study evaluated the antimicrobial effects of natural phenolic compounds extracted from vegetables, fruits, herbs and spices. The main objective was to determine the lowest concentration of phenolics to inhibit the visible growth of the pathogenic bacteria which is defined as the minimum inhibitory concentration (MIC). Some of the most common Gram-positive and Gram-negative foodborne pathogens were treated with several natural phenolic compounds. Concentrations of 5, 10, 15, and 20 ppm (pH 5-6) of each compound were evaluated by broth micro-dilution method and the MICs were determined by using official density (OD) assay. The results demonstrated that the phenolic compounds have varying antimicrobial activities against foodborne pathogens. Natural sources of phenolic compounds contain major antibacterial components and have great potential to be used as natural antimicrobials and food preservatives.

The microbiological testing of foods is a well-established science. Due to the severity of foodborne pathogen illnesses, the widespread use and implementation of rapid detection methods in food testing labs is increasingly important. The first step for successful testing is sampling. Surfactants have been highly used in food microbiology, but there is not much, if any, published research about the use of fatty alcohols and chemical dispersants as aids in microbial separation. The microbial extraction efficiency of Escherichia coli K12 and Listeria innocua from hot dogs, spinach, and milk was measured using chemical additives (surfactants, fatty alcohols, and a chemical dispersant) in a buffer solution. Dry matter content was calculated using the oven method to determine how clean the sample was at the end of processing. Tween 80 at 0.01% was found to be the most effective additive for microbial recovery for each food matrix examined. The addition of fatty alcohols to surfactants also showed much promise in aiding separation as well as in minimizing dry matter in the final solution. However, the use of Buffered Peptone Water as the diluting agent resulted in very high recovery percentages without the need for additives.

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.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Anelise_Bravo.pdf: 334250 bytes, checksum: 202476b6e47db168114279f75ea259d0 (MD5) Previous issue date: 2017-07-20 / Sem bolsa / A mussarela de leite de búfala, principal tipo de queijo obtido a partir desse leite no Brasil, é um produto novo no mercado, com alta aceitação pelos consumidores e excelentes perspectivas de comércio. O queijo é um alimento rico em nutrientes, o que favorece a proliferação de micro-organismos que podem provocar toxi-infecções ou intoxicações nos consumidores. A gastrenterite estafilocócica é causada pela ingestão de alimentos que contenham enterotoxinas produzidas por Staphylococcus coagulase positiva. Um problema que pode dificultar a eliminação de microorganismos indesejáveis na indústria de alimentos é a formação de biofilme. O objetivo deste estudo foi determinar o efeito da formação de biofilme por Staphylococcus coagulase positiva (SCP) isolados de queijo mussarela de búfala sobre a sensibilidade a sanitizantes. A partir de contagens de SCP realizadas em 50 amostras de queijo mussarela de búfala foram obtidos isolados, que foram comparados entre si por rep-PCR e identificados bioquimicamente e por multiplex PCR. As cepas distintas foram testadas quanto a formação de biofilme em placas de microtitulação. As cepas forte formadoras e uma não formadora de biofilme foram testadas em superfícies de polietileno de alta densidade, aço inoxidável e vidro. Também foram testadas quanto à sensibilidade ao hipoclorito de sódio e ao iodo após a formação do biofilme. Vinte amostras de queijo albergavam SCP, entretanto as contagens estavam dentro dos limites estabelecidos pela legislação brasileira. A rep-PCR mostrou que cada uma das amostras que estavam contaminadas apresentava uma única cepa, as quais foram identificadas como S. aureus. Dois isolados foram classificados como forte formadores de biofilme, sete como moderados formadores, dez fracos formadores e um como não formador de biofilme. As duas cepas forte formadoras produziram biofilme nas três superfícies testadas. A aplicação dos sanitizantes hipoclorito de sódio e iodo promoveu uma redução das populações bacterianas de aproximadamente 2 log em todas as superfícies, tanto das cepas formadoras de biofilme como da não formadora. Embora as cepas formadoras de biofilme não sejam mais resistentes aos sanitizantes hipoclorito de sódio e iodo do que as não formadoras, elas atingem maiores concentrações no biofilme, o que resulta em maiores populações bacterianas remanescentes após a aplicação dos sanitizantes. / The Buffalo milk mozzarella cheese, main type of chesse obtained from this milk in Brazil, is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the proliferation of microorganisms that can cause food toxi-infections in the consumer. The staphylococcal gastro-enteritis is caused by ingestion of food containing enterotoxins produced by coagulase-positive Staphylococcus. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by coagulase-positive (CPS) Staphylococcus isolated from buffalo mozzarella cheese on sensitivity to sanitizers. From CPS counts carried out on 50 samples of buffalo mozzarella cheese were obtained isolates, which were compared by rep-PCR and biochemically identified and by multiplex PCR. The distinct strains were tested for biofilm formation in microtiter plates. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. Twenty samples of cheese harbor CPS, however the counts were within the limits established by Brazilian legislation. Rep-PCR showed that each of the samples that were contaminated had a single strain, which was identified as S. aureus. Two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers.