Les bactéries pathogènes Escherichia coli entérohémorrhagiques (EHEC) O157:H7 causent des toxi-infections sévères chez l’humain. Les biofilms des EHEC rendent difficile leur contrôle dans les environnements favorisant leur persistance. Certaines souches O157:H7 dont celle de référence Sakai, possèdent une capacité accrue à s'autoagréger et former des biofilms. L’étude a visé à identifier les gènes impliqués dans l’autoagrégation et la formation de biofilm chez la souche Sakai, et vérifier l’association des facteurs identifiés avec d’autres EHEC O157:H7 du même phénotype. Avec une banque de mutants Tn10 de la souche Sakai, des mutants non-autoagglutinants et non-formateurs de biofilms des gènes csgB et csgG furent sélectionnés et caractérisés. Ces mutants formaient significativement moins de biofilms et d´autoagrégats que Sakai, et ne produisaient plus curli. La complémentation des mutants restaurait le phénotype sauvage. De plus, des gènes responsables de la biogenèse de curli csgA, csgB et csgG étaient significativement surexprimés chez Sakai, comparativement avec la souche EDL933 non-autoagglutinant, qui forme moins de biofilm. Parmi les souches d’E. coli 0157:H7 on distingue deux groupes d’isolats: Sakai-like et EDL933-like selon leur production de curli, et leur capacité à former des autoagrégats et biofilms. Nos résultats suggèrent qu’une surproduction de fibres de curli dans un sous-ensemble de souches O157:H7 pourrait être responsable de leur phénotype particulier d'autoagrégation et de formation de biofilms forts. Le projet permet de mieux cerner le mécanisme de formation de biofilm dans EHEC et renforce l’hypothèse que le curli est une cible intéressante pour contrer la persistance des EHEC en environnements naturels et industriels. / Enterohemorrhagic E. coli (EHEC) O157:H7 is an important foodborne pathogen that causes severe toxi-infections in humans. These bacteria have a higher capacity to form biofilm, impeding the control of the contamination in different environments and allowing their persistence. Some E. coli O157:H7 strains, including the Sakai reference strain, display a distinctive ability to autoaggregate and form strong biofilms. The aim of this work was to identify the genes involved in autoaggregation and biofilm formation in Sakai strain, and to verify the association between identified factors and the same phenotype in other EHEC O157: H7. We found that csgA, csgB and csgG curli genes were significantly overexpressed in strain Sakai compared to strain EDL933, a low biofilm-former and non-autoaglutinating strain. Sakai csgB and csgG Tn10 mutants formed significantly less biofilm and autoaggregation than the wild-type strain, and lost the curli-producing phenotype. Complementation restored the strong autoaggregation and biofilm formation phenotype, and the curliated morphotype of Sakai. In addition, E. coli O157: H7 isolates tested for curli formation, Sakai-like strains were curli-producing, whereas EDL933-like strains were non-curliated. These results suggest that overproduction of extracellular curli fibers in a subset of E. coli O157: H7 strains may be responsible for their particular phenotype of autoaggregation and strong biofilm formation. The project provides a better understanding of the mechanism of biofilm formation in EHEC, as well as reinforcing the hypothesis that curli fibers are an attractive target to counter the persistence of these bacteria in natural and industrial environments.

Within the last decade, pork consumption has steadily increased and continues to be the most consumed meat globally. However, pathogenic bacterial strains resistant to antibiotics have also been increasingly found in pig farms, animals, and the environment. Bacterial food poisoning cases due to Salmonella spp. and Escherichia coli (E. coli) O157:H7 appear to be linked with a variety of pork products. The meat industry has recognized that research is needed to combat the multi-drug resistance in foodborne pathogens with alternative methods of control. This study evaluated the effects of both E. coli- and Salmonella-specific lytic bacteriophages and lactic acid (LA) on E. coli O157:H7, Salmonella Enteritidis, Salmonella Montevideo, and Salmonella Heidelberg growth in raw pork loins ready for marination. The efficacy of the treatments was determined after 1 h of application and marination. Lytic bacteriophage 5% significantly (PSalmonella spp. population by 2.30 log CFU/cm2 when compared with the initial surface attachment. Moreover, the combined treatment of LA 2.5% + phage 5% significantly (PSalmonella population by 2.35 log CFU/cm2 after 1 hour of attachment. In the post-tenderization surface samples, the combination of both phage and LA showed (PP>0.05) when analyzing the translocation of Salmonella spp. on pork loins. Similar treatment efficacy results were observed in the application of E. coli O157:H7 on pork. Following antimicrobial treatments, both control and treated loin samples were enumerated after 1 h at 4°C. Both the lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% had a significant reduction of E. coli O157:H7 on surface attachment after 1 h of treatment application. Lytic bacteriophage 5% and Lactic acid 2.5% significantly (P < 0.05) reduced the surface bacterial population by 1.89 log CFU/cm2. Lytic bacteriophage 5% alone significantly (P < 0.05) reduced the surface bacterial population by 1.90 log CFU/cm2 when compared with the initial surface attachment groups. Moreover, in the post-tenderization surface samples, lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% were the only treatments that had a significant reduction (P < 0.05) when compared with the control group. Interestingly, lactic acid 2.5% was the only treatment that had a significant reduction (P < 0.05) of 0.76 log CFU/cm2 when analyzing the translocation of pathogens on pork chops.

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Grape seed extract (GSE) and peanut skin extract (PSE) are waste products in the wine and peanut industries. Both extracts have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. A subcategory of polyphenol is procyanidin, which can be divided into two types, type A and type B. Type A (PSE), contains two single bonds connecting the phenolic groups while type B (GSE), contains one single bond connecting the phenolic groups. The minimum inhibitory concentration (MIC) of the two extracts was evaluated for their antimicrobial effect on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium using the pour plate method. GSE was found to have a significantly lower MIC (p ≤ 0.05) than PSE for L. monocytogenes (GSE=60.60ppm, PSE=not found), S. aureus (GSE=38.63ppm, PSE=51.36ppm), and S. Typhimurium (GSE=45.73ppm, PSE=60.60ppm). There was no significant difference in inhibition of E. coli O157:H7 (GSE=47.44ppm, PSE=51.13ppm). Since GSE, contributed to greater pathogen inhibition, its extract was fractionated into monomer and oligomers components. Growth curves of all four pathogens inoculated in the monomer and oligomer fractions were compared using the BioScreen method. Oligomers inhibited growth of L. monocytogenes, S. aureus, and E. coli O157:H7 while monomers inhibited growth of S. Typhimurium. These results indicate that an extract with type B procyanidins that are high in oligomers may be more effective as antimicrobials. Type B procyanidins have also been shown to prevent bacterial adhesion, as is the case with urinary tract infections, and may aid in the prevention of biofilms. / Master of Science in Life Sciences

Food-borne illness outbreaks are occasionally associated with fresh-vegetable consumption, in part due to lack of a microbial inactivation step before consumption. Raw manure or improperly composted manure applied as soil amendments is an established source of pathogenic bacterial contamination. However, less is known about whether such soil amendments could serve as a source of transmission of antibiotic-resistant bacteria (ARB) or antibiotic-resistance genes (ARGs) via fresh produce. As such knowledge is developing, it is useful to identify strategies for mitigating ARGs and ARB on vegetable surfaces, especially those that are synergistic with known benefits in terms of general pathogen reduction on fresh produce. Sanitizers play an important role in post-harvest processing of vegetables, especially in terms of disinfecting the wash water and preventing cross-contamination. Further, temperature and time of storage of vegetables are critical to prevent the growth of microorganisms. To provide a background inoculum representing potential pre-harvest carryover of ARB and ARGs, carrots or romaine lettuce leaves were dipped in a slurry derived from composted manure from dairy cows previously dosed with antibiotics and further inoculated with multi-drug resistant E. coli O157:H7, a human pathogen, and a spoilage-associated and opportunistic pathogenic strain of Pseudomonas aeruginosa. Inoculated carrots (n=3, 25 g) were washed with water containing different sanitizers (sodium hypochlorite or peroxyacetic acid) or unwashed (control), packaged and stored at 10ºC for 7d or 2ºC for up to 60 d. Inoculated lettuce leaves (n=3, 100 g) were washed with sodium hypochlorite, packaged in modified atmosphere conditions (98% nitrogen), irradiated (1.0 kGy) and subsequently stored at 4ºC for 14 d. The effect of post-harvest treatment were compared at various times by enumeration on selective media. In addition, cultureindependent techniques were also performed to determine changes to the surficial carrot and lettuce microbiota by sequencing bacterial 16S rRNA gene amplicons. The effect of post-harvest treatments on the types and relative abundance of ARGs, also known as the “resistome,” were profiled by shotgun metagenomic sequencing and qPCR. Addition of a sanitizer during wash, storage temperature, and duration of storage affected the bacterial community structures on carrots, represented by the weighted Unifrac distance matrices (ANOSIM, R=0.465). Storage of sanitizer-washed carrots at 10ºC was associated with an increase in relative abundance of Pseudomonadaceae compared to 2ºC storage for 7 d (Wilcoxon, p<0.05). Increase in storage temperature from 2ºC (optimum) to 10ºC (temperature abuse) of sanitizer-washed carrots resulted in enrichment of ARGs conferring resistance to the following antibiotic classes: multidrug, peptide, polymyxin, quinolone, triclosan, aminoglycoside, bacitracin, β-lactam, and fosfomycin. Irradiation resulted in significant reductions (~3.5 log CFU/g) of inoculated antibiotic-resistant E. coli O157:H7 and Pseudomonas sp. on lettuce surfaces (ANOVA, p<0.05). The lettuce resistome, represented by the Bray-Curtis similarity of ARG occurrence, was affected by irradiation (ANOSIM, R=0.406). Irradiation of lettuce followed by 14 d of storage at 4ºC resulted in 2-4-fold reductions in relative abundance of ARGs encoding resistance to the following antibiotic classes: triclosan, quinolones, multidrug, polymyxin and β-lactam (Wilcoxon, p<0.05). No additional increase or reduction of the tet(A) gene present on inoculated P. aeruginosa was evident after 14d storage at 4ºC on irradiated samples. Results of this study suggest that inclusion of a sanitizer in wash water, irradiation, and storage at optimum refrigerated temperatures may offer effective strategies to combat proliferation of antibiotic resistant bacteria and antibiotic resistance genes on fresh produce. Further research is needed develop interventions that can mitigate tet(A) and other ARGs on produce that were not significantly reduced by irradiation. This study will guide future research on microbiome and metagenome of processed produce and assessment of critical control points to reduce the risk of antibiotic resistance from farm-to-fork. / PHD / Post-harvest interventions; such as washing, irradiation and cold storage, are employed to provide safe and wholesome fresh vegetables to consumers. Washing of vegetables in water that includes a sanitizing agent, such as chlorine or peroxyacetic acid (POAA), removes soil from the surface, reduces the bacteria in wash water and prevent cross-contamination between vegetables. It has an additional benefit to reduce microorganisms on produce surfaces that may cause the vegetables to spoil or result in illness in humans. Low temperature storage of produce, usually 0-5ºC, decreases the respiration rate of vegetables and reduces growth of microorganisms during storage. Some of the spoilage and/or pathogenic bacteria may also be antibiotic-resistant, which are commonly termed as antibiotic-resistant bacteria (ARB). Antibiotic resistance is a significant public health concern that leads to ineffective medical treatments, prolonged duration of illnesses and increased hospitalization costs. Antibiotic resistance is encoded by genes that confer resistance to wide range of antibiotic classes, including antibiotics used to treat human illnesses. These genes are termed as antibiotic resistance genes (ARGs). In this study we examined the effect of three common post-harvest interventions, washing with sanitizers, gamma irradiation, and cold storage to reduce antibiotic-resistant bacterial pathogens and antibiotic-resistant spoilage bacteria on carrots and lettuce. Storage temperature, inclusion of sanitizer in wash water, and length of chilled storage significantly influenced the diversity of bacteria found on carrot surface. Inclusion of either sanitizer in the wash water significantly reduced the populations of antibiotic-resistant E. coli O157:H7 (a pathogenic bacterium that causes a dangerous form of gastrointestinal illness) and Pseudomonas sp. (a bacterial species that commonly causes food spoilage). Storage at recommended temperature (2ºC) did not allow these bacteria to regrow and also reduced total ARGs on carrot surfaces. Washing of lettuce with sodium hypochlorite followed by irradiation (1.0 kGy) and storage at recommended temperature (4ºC) were effective in reducing the populations of antibiotic-resistant E. coli O157:H7 and Pseudomonas sp., and additionally reduced the number of some ARGs conferring resistance to select classes of antibiotics, including triclosan, quinolones, multidrug, polymyxin and β-lactam antibiotics on the lettuce surface. A novelty of this research is that it employed new, cutting-edge “metagenomic” DNA sequencing technique to identify and track antibiotic resistance through the various post-harvest interventions. Overall results of this research suggest that inclusion of sanitizer in wash water for fresh produce, followed by storage at refrigerated temperatures below 4ºC may reduce the risk posed by antibiotic resistant bacteria and antibiotic resistance genes on produce.

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.

Master of Science / Food Science Institute, Animal Science and Industry / Daniel Y.C. Fung / Within the United States, it has been estimated that 60 deaths and 73,000 illnesses are caused by Escherichia coli O157:H7 infection annually (Gavin et al., 2004). Multiple effects have been known to occur with the onset of infection from E. coli O157:H7 in which some of these can become life-threatening. Escherichia coli O157:H7 is defined as a Shiga-toxin-producing E. coli strain (STEC). This microbial pathogen is a gram-negative bacillus organism that is motile, non-sorbitol fermenting, and β-glucuronidase negative. The infectious dose of E. coli O157:H7 can be as low as ten cells (Food and Drug Administration, 2009). Consumption of contaminated food, mainly undercooked ground beef and non or incorrectly pasteurized milk, are the primary sources of E. coli O157:H7 infection in human. Cattle, in particular, are considered chief asymptomatic reservoirs for this pathogen. Carried in their gut, feces, and milk, cattle carry this Shiga toxin-producing E. coli in ranges from 10[superscript]2 to 10[superscript]5 CFU/g. Although colonized with E. coli O157:H7, cattle and other ruminants show no adverse side effects from the pathogenic bacteria. There is also a difference in the prevalence of this pathogen between human and cattle. There has been a low incidence of illness caused by E. coli O157:H7 in humans when compared to the high prevalence of E. coli 057:H7 found in cattle and their environment. It has been discovered, through population genetic analysis, that E. coli O157:H7 and other O157:H- isolates make up a clone complex. In spite of the clonal nature of E. coli O157:H7 and other O157:H[superscript]- isolates, there are significant characteristics showing variability between the clone complex. These variability aspects can possibly account for the rapid divergence of E. coli strains including the recently discovered divergence of E. coli O157:H7 in to two separate lineages. Other possible reasons for a non-linear relationship between cattle prevalence and human infection include diversity of the Shiga Toxin-Encoding bacteriophage and receptors in cattle verses human, and finally the difference between the production of Locus of Enterocyte Effacement (LEE) in both human and cattle lineages.

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.