Afin de stabiliser le microbiote intestinal et prévenir ou traiter les infections entériques, il est suggéré depuis des décennies d’utiliser certaines bactéries lactiques dites ‘probiotiques’. La consommation de ces bactéries, qui sont des composants normaux du microbiote intestinal, aurait des effets bénéfiques sur la santé. Cependant, leur éventuel rôle prophylactique ou thérapeutique n’a été que très peu étudié. Dans une première partie, cinq bifidobactéries probiotiques isolées de fèces de nouveaux-nés ont été sélectionnées et caractérisées. Parmi celles-ci, une souche de Bifidobacterium thermacidophilum (RBL71) démontrant une forte résistance aux conditions retrouvées dans le tractus intestinal, une forte adhésion aux cellules intestinales Caco-2 et une inhibition de l’adhésion d’Escherichia coli O157:H7 (50%) aux cellules Caco-2 a été administrée par voie orale à des souris BALB/c. Ce gavage probiotique avant infection à E. coli O157:H7 a diminué les comptes fécaux de ce pathogène ainsi que les dommages histologiques intestinaux comparativement au groupe témoin. Une plus forte production d’anticorps spécifiques anti-E. coli O157:H7 a également été détectée chez les souris recevant la souche probiotique. Dans une seconde partie, l’efficacité de trois autres souches de bifidobactéries a été vérifiée contre des entéropathogènes viraux. Une souche de B. thermophilum (RBL67) démontrant la plus forte inhibition de l’attachement du rotavirus (98%) aux cellules intestinales Caco-2 et HT-29 a été administrée par voie orale à des souris néonatales CD-1 infectées par un rotavirus. La concentration de virus dans le contenu intestinal était significativement moins élevée à 48 heures post-infection chez le groupe recevant des probiotiques avant infection par rapport au groupe témoin. De plus, la durée de la diarrhée a été plus courte et une production d’anticorps spécifiques anti-rotavirus a été détectée chez les souris recevant des probiotiques avant l’infection. Ces résultats suggèrent que la souche RBL67 pourrait avoir un impact positif sur l’évolution des infections causées par des pathogènes viraux invasifs tel que rotavirus et que la souche RBL71 pourrait ainsi jouer un rôle dans la prévention ou le traitement d’infections entériques aux pathogènes bactériens non invasifs tel que E. coli O157:H7. Dans ces deux cas, l’inhibition de l’adhésion du pathogène serait le mécanisme d’action le plus probable. Cette démonstration de l’activité de ces nouvelles souches de bifidobactéries d’origine humaine contre E. coli O157:H7 et rotavirus, en interférant sur le mécanisme d’infection de ces entéropathogènes, soutient leur utilisation potentielle chez l’humain pour prévenir les infections entériques transmises par voie orale. / For decades, the use of certain lactic acid bacteria as so-called probiotics has been suggested in order to stabilize the intestinal microbiota and thus prevent or treat enteric infections. Consumption of these bacteria, which are normal components of human intestinal microbiota, is reputed to be beneficial to health. However, their possible role as therapeutic or prophylactic agents has been studied very little. Five probiotic bifidobacteria isolated from the feces of newborn infants were first selected and characterized. Among these, a strain of Bifidobacterium thermacidophilum (called RBL71) demonstrating strong resistance to the conditions prevailing in the digestive tract, strong adhesion to Caco-2 intestinal cells and inhibition of the adhesion of Escherichia coli O157:H7 (50%) to Caco-2 cells was administered via the oral route to BALB/c mice. Mice thus treated before challenge had reduced fecal counts of E. coli O157:H7 and less intestinal histological damage than the control group. Greater production of O157:H7-specific antibody was detected in mice receiving the probiotic. In a second study, the effectiveness of three other strains of bifidobacteria against viral enteropathogens was examined. A strain of B. thermophilum (called RBL67) demonstrating the strongest inhibition (98%) of rotavirus attachment to Caco-2 and HT-29 intestinal cells was administered via the oral route to neonatal CD-1 mice infected with rotavirus. The viral concentration of the intestinal contents 48 hours after infection was significantly lower in the probiotic-treated group than in the control group. In addition, the diarrhea was of shorter duration and rotavirus-specific antibody production was detected in the mice receiving the probiotic before infection. These results suggest that strain RBL67 has a positive impact on the evolution of infections by invasive viral pathogens such as rotavirus and that strain RBL71 could thus have a role to play in the prevention or treatment of enteric infections by non-invasive bacterial pathogens such as E. coli O157:H7. In both cases, inhibition of adhesion of the pathogen seems to be a plausible mechanism of action. This demonstration of the activities of these new bifidobacterial strains of human origin against E. coli O157:H7 and rotavirus suggests their potential for interfering with the mechanism of infection of enteropathogens and supports their use in humans as possible agents for preventing enteric infections transmitted by the oral route.

O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

El consum de fruites fresques tallades i sucs de fruites no pasteuritzats s'haincrementat significativament en els darrers anys degut a la creixent demanda deproductes sans de baix contingut calòric i característiques similars a les del productefresc. Tot i això, aquests productes poden contaminar-se amb microorganismespatògens o deterioratius per una inadequada manipulació o emmagatzematge, donantlloc a riscs de malalties microbianes i deteriorament del producte. De fet, el nombrede brots i casos de malalties causades pel consum d'aquests productes ha augmentatnotablement, motiu pel qual l'ús de composts naturals que garanteixin la seguretat iqualitat de les fruites fresques tallades i sucs de fruites no pasteuritzats haaugmentat. L'objectiu principal d'aquesta recerca ha estat avaluar l'efectivitat dediferents antimicrobians per a controlar microorganismes patògens tals com Listeriamonocytogenes, Salmonella Enteritidis i Escherichia coli O157:H7 i allargar la vidaútil de fruites fresques tallades. Inicialment les concentracions mínimes inhibitòries(CMI) i bactericides (CMB) d'àcid màlic i diferents olis essencials (EOs) i els seuscompostos actius van ser determinats en sucs de poma, pera i meló. Desprésaquestes substàncies han estat aplicades en combinació amb compostsestabilitzadors de la qualitat directament per immersió o indirectament a través derecobriments comestibles a base d'alginat a pomes, peres i melons frescs tallats per agarantir la seva seguretat i qualitat. Els sucs de poma, pera i meló van ser inhibitorisper a L. monocytogenes, S. Enteritidis i E. coli O157:H7 a 5ºC, no obstant a 20 i35ºC concentracions mínimes d'àcid màlic de 0,2% per a L. monocytogenes i S.Enteritidis i 0,4% per a E. coli O157:H7 van ser requerides per a inhibir els seuscreixements en suc de meló. En general, concentracions d'àcid màlic de 2% en sucsde poma i pera i de 2,5% en suc de meló van ser requerits per a reduir les poblacionsde L. monocytogenes, S. Enteritidis i E. coli O157:H7 en més de 5 cicles log a 5ºC,mentre a 20 i 35ºC concentracions més baixes van ser suficients per a aconseguiraquest efecte. D'altra banda, els olis essencials de canyella, clan, herba de llimona ipalmarosa així com també els seus compostos actius van mostrar activitatantimicrobiana sobre els microorganismes patogènics però en diferents graus. Aixíl'efectivitat d'aquestes substàncies va dependre del tipus de microorganismes, tipusde suc i concentració de la substància. Una combinació d'àcid màlic al 2,5%, Nacetil-L-cisteina a l'1% glutatione a l'1% i lactat de calci a l'1% aplicada perimmersió va reduir les poblacions de L. monocytogenes i S. Enteritidis inoculades enpomes i peres fresques tallades en més de 5 cicles log el mateix dia de la preparacióde les mostres (t = 0 dies), mentre que la població de E. coli O157:H7 va mostraruna major àcid-resistència, aconseguint aquesta reducció a partir dels 3 i 14 dies enaquestes fruites respectivament. A més a més, aquesta combinació de substàncies vaoriginar una disminució en la tasa de creixement i una prolongació de la fase lag deles poblacions d'aerobis mesòfils, psicròfils i floridures i llevadures, allargant lavida útil des d'un punt de vista microbiològic fins a 23 dies en peces de poma i permés de 30 dies en peces de pera. Així mateix, un menor consum d'O2 i una menorproducció de CO2, etilé i etanol, com també un millor manteniment de la textura i elcolor foren aconseguits amb l'ús d'aquesta combinació de substàncies, aconseguintaixí un allargament de la vida útil fisicoquímica. D'altra banda, la incorporaciód'àcid màlic junt amb composts estabilitzadors de la qualitat en recobrimentcomestibles a base d'alginat va ser efectiva per a reduir les poblacions d'E. coliO157:H7 i S. Enteritidis inoculades en peces de poma i meló així com també per ainhibir el creixement de la flora nativa, tot i això un major efecte sobre aquellsmicroorganismes es va aconseguir quan ambdós àcids màlic i EOs o els seusprincipals composts actius van ser incorporats en els recobriments, mostrant ser l'oliessencial d'herba de llimona la substància antimicrobiana més efectiva. Aquestefecte antimicrobià fou a més a més potenciat amb l'augment de les concentracionsd'EOs. Una extensió de la vida útil microbiològica de peces de poma i meló foutambé aconseguida amb la incorporació d'aquestes substàncies, no obstant algunescaracterístiques fisicoquímiques i sensorials de les fruites com textura, color, olor igust van ser afectades. Els resultats demostraven que l'ús d'antimicrobians naturalsper immersió o a través de la seva incorporació en recobriments comestibles a based'alginat és una bona alternativa per a garantir la seguretat i qualitat de fruitesfresques tallades. / El consumo de frutas frescas cortadas y zumos de frutas no pasteurizadosha incrementado significativamente en los últimos años debido a la crecientedemanda de productos sanos con bajo contenido calórico y características similaresa las del producto fresco. No obstante, estos productos pueden contaminarse conmicroorganismos patógenos o deteriorativos por una inapropiada manipulación oalmacenamiento, dando lugar a riesgos de enfermedades microbianas y deterioro delproducto. De hecho, el número de brotes y casos de enfermedades causadas porconsumo de esos productos ha aumentado notablemente, razón por la cual el uso decompuestos naturales que garanticen la inocuidad y calidad de las frutas frescascortadas y zumos de frutas no pasteurizados ha aumentado. El objetivo principal deesta investigación fue evaluar la efectividad de diferentes antimicrobianos paracontrolar microorganismos patógenos tales como Listeria monocytogenes,Salmonella Enteritidis y Escherichia coli O157:H7 y extender la vida útil de frutasfrescas cortadas. Primeramente se determinaron las concentraciones mínimasinhibitorias (CMI) y bactericidas (CMB) de ácido málico y diferentes aceitesesenciales (EOs) y sus compuestos activos en zumos de manzana, pera y melón.Después, esas sustancias fueron aplicadas en combinación con compuestosestabilizadores de la calidad directamente por inmersión o indirectamente a través derecubrimientos comestibles a base de alginato a manzanas, peras y melones frescoscortados para garantizar su inocuidad y calidad. Los zumos de manzana, pera ymelón fueron inhibitorios para L. monocytogenes, S. Enteritidis y E. coli O157:H7 a5ºC, sin embargo a 20 y 35ºC, se necesitaron concentraciones mínimas de ácidomálico de 0,2% para L. monocytogenes y S. Enteritidis y 0,4% para E. coli O157:H7para inhibir su crecimiento en zumo de melón. En general, concentraciones de ácidomálico de 2% en zumos de manzana y pera y 2,5% en zumo de melón fueronrequeridas para reducir las poblaciones de L. monocytogenes, S. Enteritidis y E. coliO157:H7 en más de 5 ciclos log a 5ºC, mientras a 20 y 35ºC concentraciones másbajas fueron suficientes para alcanzar ese efecto. Por otra parte, los aceitesesenciales de canela, clavo, hierba de limón y palmarosa así como también suscompuestos activos mostraron actividad antimicrobiana sobre los microorganismospatogénicos pero en diferentes grados. Así la efectividad de esas sustancias dependiódel tipo de microorganismo, tipo de zumo y concentración de la sustancia. Unacombinación de ácido málico al 2,5%, N-acetíl-L-cisteína al 1% glutatione al 1% ylactato de calcio al 1% aplicada por inmersión redujo las poblaciones de L.monocytogenes y S. Enteritidis inoculadas en manzanas y peras frescas cortadas enmás de 5 ciclos log el mismo día de la preparación de las muestras (t = 0 días),mientras que la población de E. coli O157:H7 mostró una mayor ácido-resistencia,alcanzando esa reducción a partir de los 3 y 14 días en esas frutas respectivamente.Además esa combinación de sustancias causó una disminución en la tasa decrecimiento y una prolongación de la fase lag de las poblaciones de aerobiosmesófilos, psicrófilos y mohos y levaduras, extendiendo la vida útil desde un puntode vista microbiológico hasta 23 días en piezas de manzanas y por más de 30 días enpiezas de peras. Así mismo, un menor consumo de O2 y una menor producción deCO2, etileno y etanol, así como también un mejor mantenimiento de la textura y elcolor fueron logradas con el uso de esa combinación de sustancias, alcanzándose asíuna extensión de la vida útil fisicoquímica. Por otra parte la incorporación de ácidomálico junto con compuestos estabilizadores de la calidad en recubrimientoscomestibles a base de alginato fue efectiva para reducir las poblaciones de E. coliO157:H7 y S. Enteritidis inoculadas en piezas de manzana y melón así comotambién para inhibir el crecimiento de la flora nativa, sin embargo un mayor efectosobre esos microorganismos fue logrado cuando ambos ácido málico y EOs o susprincipales compuestos activos fueron incorporados en los recubrimientos,mostrando ser el aceite esencial de hierba de limón la sustancia antimicrobiana másefectiva. Este efecto antimicrobiano fue además potenciado con el aumento de lasconcentraciones de EOs. Una extensión de la vida útil microbiológica de piezas demanzana y melón también fue alcanzada con la incorporación de estas sustancias,sin embargo algunas características fisicoquímicas y sensoriales de las frutas comotextura, color, olor y sabor fueron afectadas. Los resultados demostraron que el usode antimicrobianos naturales por inmersión o a través de su incorporación enrecubrimientos comestibles a base de alginato es una buena alternativa paragarantizar la inocuidad y calidad de frutas frescas cortadas. / The consumption of fresh-cut fruits and unpasteurized juices hassubstantially risen over the last years mostly due to the increasing demand of healthyfood with low caloric contents and fresh-like characteristics. Nonetheless, thoseproducts can contaminate with pathogenic and deteriorative microorganisms as aconsequence of inappropriate manipulation and storage conditions, resulting in risksof microbial diseases and spoilage of the product. In fact, the number of outbreaksand cases of illness caused by consumption of fresh-cut fruits and unpasteurizedjuices has notably increased reason by which, the use of natural compounds thatassure the safety and quality of fresh-cut fruits and unpasteurized fruit juices hasincreased. To evaluate the effectiveness of different antimicrobials to controlpathogenic microorganisms such as Listeria monocytogenes, Salmonella Enteritidisand Escherichia coli O157:H7 and extend the shelf-life of fresh-cut fruits was themain objective of this research. Minimal inhibitory (MIC) and bactericidalconcentrations (MBC) of malic acid and essential oils (EOs) and their activecompounds against those pathogens were firstly determined in apple, pear andmelon juices. Then, those substances were applied in combination with qualitystabilizing compounds directly by dipping treatments or indirectly through thealginate-based edible coatings to fresh-cut apple, pear and melon to assure theirsafety and quality. Apple, pear and melon juices were inhibitory for L.monocytogenes, S. Enteritidis and E. coli O157:H7 at 5ºC, however at 20 and 35ºCminimal concentrations of 0.2% of malic acid for L. monocytogenes and S.Enteritidis and 0.4% for E. coli O157:H7 were required to inhibit their growth inmelon juice. In general, concentrations of malic acid of 2% in apple and pear juicesand 2.5% in melon juice were necessary to reduce more than 5 log cycles of L.monocytogenes, S. Enteritidis and E. coli O157:H7 at 5ºC, whereas at 20 and 35ºClower concentrations were enough to reach that effect. On the other hand, EOs ofcinnamon, clove, lemongrass and palmarosa as well as their active compoundsshowed antimicrobial activity over pathogenic microorganisms but in differentgrade. Thus, the effectiveness of those substances depended on the microorganismtype, kind of juice and concentration of the substance, thus resulting, the essential oilof lemongrass the substance more effective to inactivate those microorganisms. Acombination of 2.5% D-L malic acid, 1% N-acetyl-L-cysteine, 1% glutathione and1% calcium lactate applied through dipping treatment reduced the populations of L.monocytogenes and S. Enteritidis inoculated in fresh-cut apples and pears in morethan 5 log cycles the same day of sample preparation (t = 0 days), whereas thepopulation of E. coli O157:H7 showed a higher acid-resistance, reaching thatreduction from 3 and 14 days in those fruits respectively. In addition, thatcombination provoked a decrease of the growth rate and prolonged the lag phase ofmesophilic aerobic, psychrophilic and yeast and molds populations, extending theshelf-life from a point of view microbiological until 23 days in apple pieces and bymore than 30 days in fresh-cut pears. Likewise, a lower consumption of O2 and alower production of CO2, ethylene and ethanol, as well as a better maintenance ofthe firmness and color were achieved with the use of that substances combination,thus reaching a physicochemical shelf-life extension. On the other hand, theincorporation of malic acid with quality stabilizing compounds into alginate-basededible coatings was effective to reduce the populations of E.coli O157:H7 and S.Enteritidis inoculated in apple and melon pieces as well as to inhibit the native floragrowth. However, a higher effect over those microorganisms was achieved whenboth malic acid and EOs or their main active compounds were added into coatings,showing the essential oil of lemongrass to be the most effective antimicrobialsubstance. That antimicrobial effect was also intensified with the increase of theEOs concentrations. An extension of the microbiological shelf-life of apple andmelon pieces was also reached with the incorporation of those substances.Nonetheless, some physicochemical and sensory characteristics of the fruits such asfirmness, color, flavour and taste were affected. The results demonstrated that theuse of natural antimicrobials by immersion or through their incorporation intoalginate-based edible coatings is a good alternative to assure the safety and qualityof the fresh-cut fruits.

The application of non-thermal processes pulsed electric fields (PEF) and cross-flow micro-filtration (CFMF) continuous to be studied with the purpose of controlling microorganisms in milk. Trends suggesting increased adoption include the study of Food Safety Objectives as a safety criterion, the promotion of sustainable processing, and the implementation of hurdle strategies. While the advance of gentle processing is counteracted by the risk of enhanced resistance due to microbial stress response, several techniques can be applied to quantitatively assess its impact. The objective of this project was to evaluate the effectiveness of microbial inactivation by PEF and CFMF at various steps of milk processing including shelf-life, its comparison with high temperature short time (HTST) pasteurization, and the quantitative assessment of the cross protection resistance to PEF of Escherichia coli O157:H7. Some differences in mesophilics inactivation were observed in milks (fat contents between 1.1% and 3.1%). Increasing the PEF inlet temperature decreased the treatment time by three or two-fold. The combination of CFMF/PEF yielded similar microbial reductions as CFMF/HTST. Higher inactivation of the coliforms was achieved in homogenized cream (12% fat) compared to non-homogenized. The linear relation between electrical conductivity and nutrient content (fat and solids content) was established. In a parallel study the PEF/CFMF sequence resulted in higher inactivation of mesophilics compared to CFMF/PEF and HTST. The shelf life was acceptable for CFMF/PEF and HTST after 7 days, while enterics and psychrotrophs grew more after PEF/CFMF, thermodurics did after HTST. The growth and stress of Escherichia coli O157:H7 in lactose containing broths was monitored by absorbance and fluorescence expression of stress reporters. Growth was explained using a secondary model, and stress response using mechanistic and probabilistic models. PEF inactivation was evaluated following the Weibull distribution after the cells reached stationary phase or maximum fluorescence expression. Similar resistances were observed within the cells grown in lactose broth at 10, 25 or 40°C, as within stressed cells (starved or cold shocked). Cells grown at 45 °C were more resistant compared to the cells grown in acid, high salt concentration while the ones grown at cold temperatures were the weakest. / Dairy Farmers of Ontario, Natural Sciences and Engineering Research Council.

O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

The production of final effluents that meet discharged requirements and guidelines remain a major challenge particularly in the developing world with the resultant problem of surface water pollution. This study assessed the physicochemical and microbiological qualities of two wastewater treatment works in the Eastern Cape Province of South Africa in terms of the prevalence of faecal coliforms and Escherichia coli O157:H7 over a five month period. All physicochemical and microbiological analyses were carried out using standard methods. Data were collected in triplicates and analysed statistically using IBM SPSS version 20.0. The ranges of some of the physicochemical parameters that complied with set guidelines include pH (6.7 – 7.6), TDS (107 – 171 mg/L), EC (168 – 266 μS/cm), Temperature (15 – 24oC), NO3- (0 – 8.2 mg/L), NO2- (0.14 – 0.71 mg/L) and PO4 (1.05 – 4.50 mg/L). Others including Turbidity (2.64 – 58.00 NTU), Free Cl (0.13 – 0.65 mg/L), DO (2.20 – 8.48 mg/L), BOD (0.13 – 6.85 mg/L) and COD (40 – 482 mg/L) did not comply with set guidelines. The microbiological parameters ranged 0 – 2.7 × 104 CFU/100 ml for FC and 0 – 9.3 × 103 for EHEC CFU/100 ml, an indication of non-compliance with set guidelines. Preliminary identification of 40 randomly selected presumptive enterohemorrhagic E. coli isolates by Gram’s staining and oxidase test shows 100% (all 40 selected isolates) to be Gram positive while 90% (36 randomly selected isolates) were oxidase negative. Statistical correlation between the physicochemical and the microbiological parameters were generally weak except in the case of free chlorine and DO where they showed inverse correlation with the microbiological parameters. The recovery of EHEC showed the inefficiency of the treatment processes to effectively inactivate the bacteria, and possibly other pathogenic bacteria that may be present in the treated wastewater. The assessment suggested the need for proper monitoring and a review of the treatment procedures used at these treatment works.

Growing consumer awareness of the health benefits associated with fruits and vegetables and demand for easy to prepare products has prompted the development of a wide variety of minimally processed fruits and vegetables. Minimally processed fruits and vegetables are often peeled, cut, or diced which compromise the produces’ natural protective barriers, exposing a nutrient rich medium and providing an ideal environment for the growth of microorganisms, including foodborne pathogens. The germination conditions of sprout vegetables consisting of relatively high temperatures and humidity, low light and abundance of nutrients are also conducive to the proliferation of foodborne pathogens. Recent outbreaks and recalls indicate additional measures are needed to improve food safety and maintain the integrity of the food industry. The objective of this research was to evaluate the efficacy of Lactic Acid Bacteria (LAB) against E. coli O157:H7, L. monocytogenes, and Salmonella spp. on apple slices and alfalfa sprouts and it’s influence on product quality. Apple slices inoculated with E. coli O157:H7, L. monocytogenes, and Salmonella spp. (each at 104 CFU/g) were treated with Lb. plantarum alone and in combination with Pediococcus acidophilus and P. pentosaceus (LPP) (107 CFU/g) while alfalfa seeds were inoculated with L. monocytogenes and Salmonella spp. (each at 101 CFU/g and 103 CFU/g) and treated with LPP (107 CFU/g). The growth of the microorganisms on the apple slices was assessed during five and seven days of storage at 4◦C and 20◦C, respectively. Growth on alfalfa seeds was reported during five days of sprouting at 20◦C. Populations of LAB were maintained between 7.0 log CFU/g and 8.0 log CFU/g throughout storage and sprouting on the sliced apples and alfalfa seeds, respectively. Although LAB had no significant effect on pathogen populations on apple slices during storage at 4°C (p > 0.05), populations were significantly different at 20°C (p < 0.05). Populations of L. monocytogenes in the presence of Lb. plantarum and LPP were 1.84 log CFU/g and 2.84 log CFU/g less than the controls after five days of storage at 20°C (p < 0.05). Populations of E. coli O157:H7 in the presence of Lb. plantarum and LPP were 1.83 log CFU/g and 1.86 log CFU/g less than the control after one and three days of storage, respectively. Finally, populations of Salmonella spp. were 0.86 log CFU/g less than populations in the absence of LPP after three days of storage. LPP had a significant effect on the growth of L. monocytogenes and Salmonella spp. on alfalfa seeds (p < 0.05). After five days of sprouting, populations of L. monocytogenes at an initial concentration of 101 CFU/g and 103 CFU/g on seeds treated with LPP were approximately 4.5 log CFU/g and 1.0 log CFU/g less than the untreated seeds, respectively. Populations of Salmonella spp. at an initial concentration of 101 CFU/g and 103 CFU/g were 1.0 log CFU/g less than the control. Overall, on apple slices the combination of Lb. plantarum with P. acidophilum and P. pentosaceus demonstrated greater efficacy than Lb. plantarum alone and reduction of L. monocytogenes by Lb. plantarum and LPP was greater than Salmonella spp. and E. coli O157:H7 on apple slices and alfalfa seeds, alike. LAB had a minimal effect on the quality of the apple slices and alfalfa seeds. LAB could be an effective strategy in reducing pathogen populations at abusive temperatures and germination conditions without influencing the quality of minimally processed fruit and vegetables.

<p>Modeling microbial inactivation has a great influence on the optimization, control and design of food processes. In the area of food safety, modeling is a valuable tool for characterizing survival curves and for supporting food safety decisions. The modeling of microbial behavior is based on the premise that the response of the microbial population to the environment factors is reproducible. And that from the past, it is possible to predict how these microorganisms would respond in other similar environments. Thus, the use of mathematical models has become an attractive and relevant tool in the food industry.</p> <p>This research provides tools to relate the inactivation of microorganisms of public health importance with processing conditions used in nonisothermal and non-thermal food processing technologies. Current models employ simple approaches that do not capture the realistic behavior of microbial inactivation. This oversight brings a number of fundamental and practical issues, such as excessive or insufficient processing, which can result in quality problems (when foods are over-processed) or safety problems (when foods are under-processed). Given these issues, there is an urgent need to develop reliable models that accurately describe the inactivation of dangerous microbial cells under more realistic processing conditions and that take into account the variability on microbial population, for instance their resistance to lethal agents. To address this urgency, this dissertation focused on mathematical models, combined mathematical tools with microbiological science to develop models that, by resembling realistic and practical processing conditions, can provide a better estimation of the efficacy of food processes. The objective of the approach is to relate the processing conditions to microbial inactivation. The development of the modeling approach went through all the phases of a modeling cycle from planning, data collection, formulation of the model approach according to the data analysis, and validation of the model under different conditions than those that the approach was developed.</p> <p>A non-linear ordinary differential equation was used to describe the inactivation curves with the hypothesis that the momentary inactivation rate is not constant and depends on the instantaneous processing conditions. The inactivation rate was related to key process parameters to describe the inactivation kinetics under more realistic processing conditions. From the solution of the non-linear ordinary differential equation and the optimization algorithm, safety inferences in the microbial response can be retrieved, such as the critical lethal variable that increases microbial inactivation. For example, for nonisothermal processes such as microwave heating, time-temperature profiles were modeled and incorporated into the inactivation rate equation. The critical temperature required to increase the microbial inactivation was obtained from the optimization analysis. For non-thermal processes, such as cold plasma, the time-varying concentration of reactive gas species was incorporated into the inactivation rate equation. The approach allowed the estimation of the critical gas concentration above which microbial inactivation becomes effective. For Pulsed Electric Fields (PEF), the energy density is the integral parameter that groups the wide range of parameters of the PEF process, such as the electric field strength, the treatment time and the electrical conductivity of the sample. The literature has shown that all of these parameters impact microbial inactivation. It has been hyphothesized that the inactivation rate is a function of the energy density and that above a threshold value significant microbial inactivation begins. </p> <p>The differential equation was solved numerically using the Runge-Kutta method (<i>ode45</i> in MATLAB ®). The<i> lsqcurvefit</i> function in MATLAB ® estimated the kinetic parameters. The approach to model microbial inactivation, whether when samples were subjected to nonisothermal or to non-thermal food processes, was validated using data published in the literature and/or in other samples and treatment conditions. The modeling approaches developed by this dissertation are expected to assist the food industry in the development and validation process to achieve the level of microbial reduction required by regulatory agencies. In addition, it is expected to assist the food industry in managing food safety systems through support food safety decision-making, such as the designation of the minimal critical parameter that may increase microbial inactivation. Finally, this dissertation will contribute in depth to the field of food safety and engineering, with the ultimate outcome of having a broad and highly positive impact on human health by ensuring the consumption of safe food products.</p>

Most bacterial infections can be correlated to contamination of consumables such as food and water. Upon contamination, boil water advisories have been ordered to ensure water is safe to consume, despite the evidence that heat-killed bacteria can induce genomic instability of exposed (intestine) and distal cells (liver and spleen). We hypothesize that exposure to components of heat-killed Escherichia coli O157:H7 will induce genomic instability within animal cells directly and indirectly exposed to these determinants. Mice were exposed to various components of dead bacteria such as DNA, RNA, protein or LPS as well as to whole heat-killed bacteria via drinking water. Here, we report that exposure to whole heat-killed bacteria and LPS resulted in significant alterations in the steady state RNA levels and in the levels of proteins involved in proliferation, DNA repair and DNA methylation. Exposure to whole heat-killed bacteria and their LPS components also leads to increased levels of DNA damage. / xiv, 132 leaves : ill. (chiefly col.) ; 29 cm