El principal objetivo fue estimar la prevalencia y caracterizar las <i>E. coli</i> O157 de ganado vacuno faenado en Argentina. Se recolectaron un total de 1622 muestras de heces y carcasas en 9 frigoríficos de exportación. Todas las muestras se sometieron a separación inmunomagnética y las cepas fueron identificadas por PCR múltiple (rfb<SUB>O157</SUB>, stx<SUB>1</SUB>, stx<SUB>2</SUB>). Se aislaron 54 STEC O157 y 48 <i>E. coli</i> O157 toxina Shiga negativas (EC O157 TSN), de las que se establecieron sus características fenotípicas, genotípicas y las variantes stx. La prevalencia promedio de STEC O157 en materia fecal fue de 4,1% y 2,6% en carcasa; mientras que para EC O157 TSN la incidencia fue de 4,7 y 2,6%; respectivamente. No se observaron diferencias significativas por el género o raza de los animales. Los terneros y vaquillonas presentaron mayores porcentajes de prevalencia de STEC O157 en heces (10,5 y 8,5%, respectivamente). Todas las STEC O157 aisladas albergaban los genes stx<SUB>2</SUB>, eae, ehxA, y fliC<SUB>H7</SUB> y sólo el 16,7% presentó el gen stx<SUB>1</SUB>. El genotipo prevalente fue el stx<SUB>2</SUB>/stx<SUB>2c(vh-a)</SUB>, que también es frecuente en los casos de SUH. Mediante XbaI-PFGE se obtuvieron 29 patrones diferentes y 11 clusters. En cinco oportunidades, las cepas de STEC O157 aisladas de las carcasas fueron idénticas a las cepas de materia fecal. También 7 cepas idénticas se aislaron de carcasas muestreadas en dos visitas consecutivas a dos frigoríficos. Cinco perfiles de fago tipo-PFGE-stx fueron coincidentes con perfiles de cepas recuperadas de SUH. Las técnicas de subtipificación molecular mostraron que las cepas EC O157 TSN presentan un origen filogenético diferente a STEC O157. Se cuantificó la resistencia ácida (RA) por 3 mecanismos y se determinó que el sistema Glutamato-dependiente proporciona mejor protección en desafío ácido. Las EC O157 TSN fueron, en promedio, más resistentes que las cepas STEC O157 por los sistemas descarboxilasa dependientes.

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d'un syndrome hémolytique et urémique. Une fois arrivés dans l'intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d'attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d'effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l'expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l'expression du LEE. GadE active l'expression de gadX, et GadX réprime l'expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l'expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l'expression de gadE et de gadX. Le monoxyde d'azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l'expression du LEE et active celle de gadE et de gadX. Notre étude a permis d'identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l'expression de gadE et gadX et active l'expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l'expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l'expression de gadE et donc de gadX. Un modèle de régulation intégrant l'ensemble de ces résultats est proposé. D'autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d'hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l'expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157 is a major foodborne pathogen. The bovine gut is the primary reservoir and the organism is shed in the feces, which serves as the major source of contamination. The objectives of our research on E. coli O157 were to 1) determine prevalence and characterize presence in the rectoanal mucosa of cattle, 2) rationalize preferential prevalence in the hindgut, 3) evaluate fecal prevalence and concentration in relation to carcass contamination 4) determine the efficacy of preharvest intervention strategies to reduce fecal prevalence and/or concentration in cattle. We determined that E. coli O157 isolated from the rectoanal mucosa were similar to fecal isolates. We evaluated methods of enumeration in cattle feces to identify fecal samples with > 10[superscript3] and 10[superscript4] CFU of E. coli O157/g. We observed that prevalence of E. coli O157 on carcasses was correlated with high-shedders. We identified gluconic acid as a substrate which stimulates growth of E. coli O157 in fermentations with ruminal microbial or fecal microbial inocula from cattle. This may explain the preference to persist or colonize the hindgut because gluconic acid is a component of colonic mucin. Additionally, we evaluated the effects of cattle diets (two grain types and two grain processing methods), which present different amounts of fermentable starch to the hindgut, on prevalence of E. coli O157. Dry-rolled grain-based diets reduced prevalence of E. coli O157, possibly because of increased flow of starch to the hindgut. Competitive exclusion cultures of E. coli in in vitro fermentations reduced E. coli O157 in ruminal microbial inoculum, but in fecal microbial inoculum cultures were only efficacious when gluconic acid also was added. Lastly, we evaluated a vaccine which targeted the siderophore receptor/porin proteins of E. coli O157. The vaccine reduced prevalence, the total number of days cattle tested positive, and the total number of days cattle were identified as high-shedders of E. coli O157. In summary, our research adds further knowledge to the literature about E. coli O157 in the hindgut, provides methods to identify high-shedding animals, demonstrates the importance of high-shedding animals, and offers information about potential preharvest interventions.

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.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) and enteropathogenic E. coli O127:H7 (EPEC) induce characteristic F-actin rich pedestals on infected mammalian cells. Each pathogen delivers its own translocated intimin receptor (Tir) to the host cell to act as a receptor for the bacterial outer membrane adhesin, intimin. Interaction of translocated Tir with intimin is essential for mammalian cell binding and host colonization, as well as to induce actin pedestal formation in vitro. In spite of these parallels, EHEC and EPEC Tir appear to generate actin pedestals by distinct mechanisms. Further, while the ability to form actin pedestals is a striking phenotype, the function of pedestals during infection remains unclear. To address these issues, a systematic and quantitative analysis of Tir-mediated actin assembly was conducted. We identified a three-residue Tir sequence involved in actin pedestal formation for both EHEC and EPEC, and developed evidence that the two pathogens trigger a common pathway for actin assembly. Further, the ability of these bacteria to promote actin assembly appears to promote both intimin-mediated bacterial binding in vitro and optimal colonization during experimental animal infection.

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.

Listeria monocytogenes continues to challenge the dairy industry in causing post-process contamination of cheeses. To reduce risk of contamination, it is crucial to understand the growth and survival of pathogenic bacteria in cheese products and to develop post-process mitigation strategies. This study evaluated the fate of pathogens in reduced and regular sodium Mozzarella cheese, and the potential of Supercritical Fluid Extraction with CO2 (SFE) to reduce Listeria innocua on Mozzarella and Queso Fresco. The survival of L. monocytogenes, Salmonella, and E.coli O157:H7 (2-3 log CFU/g) in reduced sodium Mozzarella (1.62%), compared to regular sodium Mozzarella cheese (2.15%) at 4ºC and 12ºC for 90 and 30 days, respectively, was evaluated. Salmonella and E. coli O157:H7 populations decreased over incubation time at both temperatures and no difference (pListeria monocytogenes population also decreased during incubation time at 4°C regardless of the sodium concentration in Mozzarella cheese. However, there was a difference in the population of L. monocytogenes for regular and reduced sodium incubated 12°C, and its populations increased 1 log CFU/g in reduced sodium Mozzarella cheese. Additionally, this study determined the bactericidal effect of SFE on the population of L. innocua, a surrogate for L. monocytogenes, in Mozzarella and Queso Fresco cheese (6 log CFU/g) treated with SFE at two pressures and temperatures (120 bar at 40°C and 150 bar at 50°C) for 30 min. SFE treatment at 120 bar, 40°C for 30 min decreased L. innocua by approximately 3.0 and 3.5 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. SFE at 150 bar and 50°C reduced L. innocua by approximately 3.78 and 5.2 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. Since SFE had a minimal effect on the physico-chemical characteristics of the cheeses assayed, the results suggest SFE might be used to reduce L. monocytogenes in cheeses without negatively impacting product quality.

No description available.

<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>

The effects of electron beam (e-beam) irradiation and sanitizers in the reduction of Escherichia coli O157:H7 and Salmonella counts and attachment prevention on spinach was studied. Survival of these pathogens in spinach was observed at multiple times and temperatures. Inoculated spinach was examined by confocal microscopy to determine attachment sites and internalization of these pathogens. To determine the effectiveness of sanitizers in reducing pathogen numbers, inoculated spinach was treated with L-lactic acid, peroxyacetic acid, calcium hypochlorite, ozone, and chlorine dioxide. Inoculated spinach was exposed to e-beam irradiation and tested for counts of both pathogens immediately after irradiation treatment to determine bacterial reduction, and at 2 day intervals over 8 days to determine effects of ionizing irradiation on pathogen survival. Respiration rates were measured on spinach exposed to e-beam. The effectiveness of e-beam irradiation on the microbiological and sensory characteristics of spinach was studied. For spinach samples stored at 4�C and 10�C for 8 days, E. coli O157:H7 survived and grew significantly in samples stored at 21�C for 24 h. Confocal microscopy images provided valuable information on the attachment sites and internalization of the pathogens on spinach. The greatest reduction by a chemical sanitizer was 55�C L-lactic acid with a 2.7 log CFU/g reduction for E. coli O157:H7 and 2.3 log CFU/g reduction for Salmonella. Each dose of e-beam irradiation significantly reduced populations of both pathogens. Respiration rates of spinach increased as irradiation treatment doses increased. Total aerobic plate counts were reduced by 2.6 and 3.2 log CFU/g at 0.7 and 1.4 kGy, respectively. Lactic acid bacteria were reduced at both doses but grew slowly over the 35 day period. Yeasts and molds were not reduced in samples exposed to 0.7 kGy whereas 1.4 kGy had significantly reduced counts. Gas compositions for samples receiving 0.7 and 1.4 kGy were significantly different than controls. Irradiation did not affect the objective color or basic taste, aromatic or mouthfeel attributes of spinach. These results suggest that low dose e-beam irradiation may be a viable tool for reducing microbial populations or eliminating E. coli O157:H7 and Salmonella from spinach with minimal product damage.