Since 1987, the Food Safety and Inspection Service (FSIS) has implemented a zero-tolerance policy for Listeria monocytogenes (Lm) in ready-to-eat (RTE) meat and poultry products, which culminated with the implementation of the Listeria rule in 2003. While researchers have extensively examined human listeriosis and its causative agent, Lm, there remained a significant gap in the current literature regarding how, singly or in combination, establishment size, RTE product type, Listeria alternative used, and FSIS district of production predict compliance with the Listeria rule. Therefore, the purpose of this quantitative study was to investigate the relationship between establishment size, RTE product type, Listeria alternative used, FSIS district of production, and compliance with the Listeria rule. The deterrence theory was used to explain the relationships and associations between variables. Archival Lm sampling data collected between 2012 and 2015 by FSIS was used to analyze the relationships. Chi-square tests showed no significant statistical relationship between establishment size, Listeria alternative used, FSIS district, and compliance, but they did show a significant association between compliance, RTE salt-cured products, and fully cooked products. Additionally, logistic regression analysis showed that the odds of an Lm-positive sample was higher for salt-cured products than for fully-cooked products. This study's findings indicate the need for a reevaluation of FSIS Listeria prevention policy, with a focus on salt-cured products. These results can influence positive social change if used in a targeted public health outreach/education program that focuses on the food safety risks associated with salt-cured products.

En Argentina, la explotación avícola se ha intensificado progresivamente hasta convertirse en industrial. Tal situación, ha conducido a mayores riesgos de contraer enfermedades de origen alimentario a través del consumo de carne contaminada. La transmisión de microorganismos patógenos en la cadena productiva se asocia con la crianza de los animales, el tratamiento en las plantas de faena y la manipulación durante la comercialización. Dentro de los patógenos alimentarios, Listeria monocytogenes es uno de los más importantes, debido a su impacto económico e interés para la salud pública. Esta bacteria crece a temperaturas de refrigeración y se adapta a diferentes condiciones de estrés. En la búsqueda de alternativas seguras e inocuas para controlar L. monocytogenes surge explorar el potencial de las bacterias ácido lácticas (BAL) endógenas de pollos parrilleros. Estas cumplen un rol fundamental en el mantenimiento de la microbiota intestinal y la homeostasis, pudiendo inhibir cepas potencialmente patógenas a través de la producción de sustancias inhibidoras tipo bacteriocinas. Dentro de las estrategias para promover su desarrollo, se encuentra el empleo de dietas basadas en probióticos u otros suplementos naturales. Hallar BAL adaptadas al ambiente intestinal que funcionen como antagonistas, es un desafío interesante por su fácil disponibilidad y bajo costo. En este Trabajo de Tesis se evaluó previamente en el Capítulo I la calidad microbiológica de distintos cortes de pollo provenientes de comercios locales. Los resultados evidenciaron la presencia de L. monocytogenes, con otras especies acompañantes incluyendo L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, L. grayi y L. murrayi. Posteriormente, en el Capítulo II se analizó la capacidad adaptativa de las cepas de L. monocytogenes en medios conteniendo 1,5 % y 3 % p/v de cloruro de sodio (NaCl). Los estudios mostraron un comportamiento heterogéneo de las cepas frente a las distintas concentraciones de NaCl. Las diferencias observadas en el recuento se atribuyeron a la variabilidad fenotípica existente dentro de la especie. En el Capítulo III se aislaron BAL en excretas provenientes de pollos sometidos a distintas dietas experimentales. Las dietas incluían suplementos basados en el probiótico B. subtilis y harina de chía (Salvia hispánica L.). Los resultados demostraron que la composición de la dieta altera de forma significativa la abundancia de BAL intestinales, con un efecto positivo tras la incorporación de la harina de chía. Finalmente, en el Capítulo IV se evaluó la capacidad antagonista de las BAL obtenidas. En este caso, se ensayaron sobrenadantes libres de células (SLC) sobre las cepas de L. monocytogenes aisladas de la carne de pollo. Los estudios indicaron que ciertos SLC presentaron antagonismo, aunque la respuesta fue específica en cada caso. Se observó una gran variabilidad en el espectro de control (efectividad) y en la actividad antagónica, con diferencias significativas entre los halos de inhibición medidos. Los datos presentados en esta Tesis, revelan que L. monocytogenes constituye un riesgo para la salud pública y que determinadas BAL nativas o autóctonas provenientes de las excretas, pueden ser útiles para su control. Estas BAL pueden resultar de interés tanto en el desarrollo de probióticos para aves, así como para la producción de sustancias antimicrobianas, de importancia en la industria alimentaria. / In Argentina, poultry farming has progressively intensified to become industrial. Such a situation has led to higher risks of contracting foodborne diseases through the consumption of contaminated meat. The transmission of pathogenic microorganisms in the production chain is associated with the rearing of animals, treatment in slaughter plants and handling during commercialization. Within the food pathogens, Listeria monocytogenes is one of the most important, due to its economic impact and interest for public health. This bacterium grows at refrigeration temperatures and adapts to different stress conditions. In the search for safe and innocuous alternatives to control L. monocytogenes, it is necessary to explore the potential of endogenous lactic acid bacteria (LAB) from broiler chickens. These play a fundamental role in the maintenance of intestinal microbiota and homeostasis, being able to inhibit potentially pathogenic strains through the production of inhibitory substances such as bacteriocins. Among the strategies to promote its development, is the use of diets based on probiotics or other natural supplements. Finding LABs adapted to the intestinal environment that function as antagonists is an interesting challenge due to their easy availability and low cost. In this Thesis Work, the microbiological quality of different cuts of chicken from local businesses was previously evaluated in Chapter I. The results showed the presence of L. monocytogenes, with other companion species including L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, L. grayi and L. murrayi. Subsequently, in Chapter II, the adaptive capacity of L. monocytogenes strains in media containing 1.5 % and 3 % p/v sodium chloride (NaCl) was analyzed. The studies showed a heterogeneous behavior of the strains against the different concentrations of NaCl. The differences observed in the count were attributed to the phenotypic variability existing within the species. In Chapter III, LAB were isolated in excreta from chickens subjected to different experimental diets. The diets included supplements based on the probiotic B. subtilis and chia flour (Salvia hispánica L.). The results showed that the composition of the diet significantly alters the abundance of intestinal LAB, with a positive effect after the incorporation of chia flour. Finally, in Chapter IV the antagonistic capacity of the LAB obtained was evaluated. In this case, cell-free supernatants (SLC) were tested on L. monocytogenes strains isolated from chicken meat. Studies indicated that certain SLCs were antagonistic, although the response was specific in each case. A great variability was observed in the control spectrum (effectiveness) and antagonistic activity, with significant differences in the measured inhibition halos. The data presented in this Thesis show that L. monocytogenes constitutes a risk to public health and that certain native or autochthonous LABs from excreta may be useful for its control. These LABs may be of interest either in the development of probiotics for poultry, as well as for the production of antimicrobial substances, of importance in the food industry.

Listeria monocytogenes é um patógeno intracelular facultativo responsável por listeriose, uma doença de origem alimentar. Dentre os 13 sorotipos de L. monocytogenes existentes, o sorotipo 4b é o mais freqüentemente isolado de humanos, enquanto os sorotipos 1/2a, 1/2b e 1/2c são mais freqüentes em alimentos e amostras ambientais. O estudo da variabilidade de virulência de quatro cepas de L. monocytogenes recentemente isoladas (1/2B - alimento; 1/2C e 4BENV - ambiente de processamento de alimento; 4BCLIN - sangue), além de duas outras cepas de casos de listeriose (P14 e P14A), foi o objetivo deste trabalho. Avaliou-se a capacidade de invasão, proliferação intracelular, citotoxicidade, disseminação intercelular, polimerização de filamentos de actina e transcrição gênica dos genes de virulência actA, plcA e plcB das cepas em modelos celulares eucarióticos MDBK, HeLa e L929. A cepa de origem alimentar 1/2B apresentou características de invasão, taxa de multiplicação (IGC), assim como a disseminação intercelular, semelhantes às cepas clínicas P14 e 4BCLIN em células HeLa. As cepas de origem ambiental (1/2C e 4BENV), apesar da baixa capacidade de invasão em todas linhagens celulares empregadas, apresentaram IGC superior às demais cepas estudadas e disseminação intercelular superior ou similar às cepas clínicas, dependendo da célula eucariótica. As cepas de origem clínica 4BCLIN, P14, P14A tiveram maiores porcentagens de invasão nas linhagens MDBK e L929, porém, em relação à população intracelular (UFC/mL), as cepas 4BCLIN e P14A foram superiores às demais. Estas últimas também foram citotóxicas às células HeLa, induzindo à liberação de LDH e à morte por necrose. Já as cepas de origem alimentar, ambiental e a cepa clínica P14 não foram citotóxicas às células HeLa e MDBK. Em relação à polimerização dos filamentos de actina não houve diferença estatística entre as cepas. Todos os genes de virulência estudados apresentaram maior número de transcritos para as cepas de origem não clínica (1/2B, 1/2C e 4BENV). Sendo assim, todas as cepas de L. monocytogenes estudadas possuem potencial patogênico, por apresentar pelo menos duas características relacionadas à virulência da espécie. Alimentos possivelmente contaminados com estas cepas representam risco à saúde pública. / Listeria monocytogenes is a facultative intracellular pathogen responsible for listeriose, a foodborne disease. Amongst the 13 serotypes of L. monocytogenes, the serotype 4b is more frequently isolated from clinical samples while the serotypes 1/2a, 1/2b and 1/2c are associated with food and food processing environment. The study of the virulence variability of four L. monocytogenes strains recently isolated (1/2B - food; 1/2C and 4BENV - food processing environment; 4BCLIN blood), as well as two strains of listeriosis (P14 and P14A) was the aim of this work. Eukaryotic cellular models HeLa, MDBK and L929 were used to evaluate the invasion capacity, intracellular proliferation, cytotoxicity, intercellular dissemination and polymerization of actin tails of L. monocytogenes strains. Gene transcription of the virulence genes actA, plcA and plcB was also conducted. The food isolate 1/2B presented invasion characteristics, multiplication index (IGC), as well as the intercellular dissemination, similar to the clinical strains P14 and 4BCLIN in HeLa cells. Environmental strains (1/2C and 4BENV), although showing a low capacity of invasion in all cellular models, had the highest IGC comparing to the other isolates. They also showed similar to or higher intercellular dissemination than the clinical ones, depending on the cell line used. The clinical strains 4BCLIN, P14, P14A presented greater invasion capacity in MDBK and L929 cells than all other isolates. However, in relation to the intracellular population (CFU/mL) the isolates 4BCLIN and P14A showed superior results. These strains were also cytotoxic to HeLa cells, inducing LDH release and death due to necrosis. The food, environmental and P14 isolates were not cytotoxic to HeLa and MDBK cells. There was no statistical difference in actin tail polymerization between the strains. The non-clinical isolates (1/2B, 1/2C and 4BENV) presented higher number of transcripts for than the clinical ones. In conclusion, all L. monocytogenes strains studied showed pathogenic potential, based on expression of at least two characteristics related to the virulence of the species. The presence of these strains in food represents public health risk.

Listeria monocytogenes é um patógeno intracelular facultativo responsável por listeriose, uma doença de origem alimentar. Dentre os 13 sorotipos de L. monocytogenes existentes, o sorotipo 4b é o mais freqüentemente isolado de humanos, enquanto os sorotipos 1/2a, 1/2b e 1/2c são mais freqüentes em alimentos e amostras ambientais. O estudo da variabilidade de virulência de quatro cepas de L. monocytogenes recentemente isoladas (1/2B - alimento; 1/2C e 4BENV - ambiente de processamento de alimento; 4BCLIN - sangue), além de duas outras cepas de casos de listeriose (P14 e P14A), foi o objetivo deste trabalho. Avaliou-se a capacidade de invasão, proliferação intracelular, citotoxicidade, disseminação intercelular, polimerização de filamentos de actina e transcrição gênica dos genes de virulência actA, plcA e plcB das cepas em modelos celulares eucarióticos MDBK, HeLa e L929. A cepa de origem alimentar 1/2B apresentou características de invasão, taxa de multiplicação (IGC), assim como a disseminação intercelular, semelhantes às cepas clínicas P14 e 4BCLIN em células HeLa. As cepas de origem ambiental (1/2C e 4BENV), apesar da baixa capacidade de invasão em todas linhagens celulares empregadas, apresentaram IGC superior às demais cepas estudadas e disseminação intercelular superior ou similar às cepas clínicas, dependendo da célula eucariótica. As cepas de origem clínica 4BCLIN, P14, P14A tiveram maiores porcentagens de invasão nas linhagens MDBK e L929, porém, em relação à população intracelular (UFC/mL), as cepas 4BCLIN e P14A foram superiores às demais. Estas últimas também foram citotóxicas às células HeLa, induzindo à liberação de LDH e à morte por necrose. Já as cepas de origem alimentar, ambiental e a cepa clínica P14 não foram citotóxicas às células HeLa e MDBK. Em relação à polimerização dos filamentos de actina não houve diferença estatística entre as cepas. Todos os genes de virulência estudados apresentaram maior número de transcritos para as cepas de origem não clínica (1/2B, 1/2C e 4BENV). Sendo assim, todas as cepas de L. monocytogenes estudadas possuem potencial patogênico, por apresentar pelo menos duas características relacionadas à virulência da espécie. Alimentos possivelmente contaminados com estas cepas representam risco à saúde pública. / Listeria monocytogenes is a facultative intracellular pathogen responsible for listeriose, a foodborne disease. Amongst the 13 serotypes of L. monocytogenes, the serotype 4b is more frequently isolated from clinical samples while the serotypes 1/2a, 1/2b and 1/2c are associated with food and food processing environment. The study of the virulence variability of four L. monocytogenes strains recently isolated (1/2B - food; 1/2C and 4BENV - food processing environment; 4BCLIN blood), as well as two strains of listeriosis (P14 and P14A) was the aim of this work. Eukaryotic cellular models HeLa, MDBK and L929 were used to evaluate the invasion capacity, intracellular proliferation, cytotoxicity, intercellular dissemination and polymerization of actin tails of L. monocytogenes strains. Gene transcription of the virulence genes actA, plcA and plcB was also conducted. The food isolate 1/2B presented invasion characteristics, multiplication index (IGC), as well as the intercellular dissemination, similar to the clinical strains P14 and 4BCLIN in HeLa cells. Environmental strains (1/2C and 4BENV), although showing a low capacity of invasion in all cellular models, had the highest IGC comparing to the other isolates. They also showed similar to or higher intercellular dissemination than the clinical ones, depending on the cell line used. The clinical strains 4BCLIN, P14, P14A presented greater invasion capacity in MDBK and L929 cells than all other isolates. However, in relation to the intracellular population (CFU/mL) the isolates 4BCLIN and P14A showed superior results. These strains were also cytotoxic to HeLa cells, inducing LDH release and death due to necrosis. The food, environmental and P14 isolates were not cytotoxic to HeLa and MDBK cells. There was no statistical difference in actin tail polymerization between the strains. The non-clinical isolates (1/2B, 1/2C and 4BENV) presented higher number of transcripts for than the clinical ones. In conclusion, all L. monocytogenes strains studied showed pathogenic potential, based on expression of at least two characteristics related to the virulence of the species. The presence of these strains in food represents public health risk.

Master of Science / Department of Anatomy and Physiology / Jishu Shi / The development of reverse genetics systems for the manipulation of viral and bacterial genomes has provided platforms for identifying virulence genes, studying pathogenesis and developing vaccines. Replication-competent vaccines (e.g., modified live virus (MLV) vaccines and replicating viral/bacterial vectors) are considered the most efficacious approach for vaccine development. We constructed replication-competent candidate vaccines for two viral diseases in pigs via reverse genetics. The first vaccine we designed is to protect against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). HP-PRRSV can cause high mortality in pigs of all ages. Vaccines to protect pigs from HP-PRRSV are not commercially available in the US. According to previous studies, the non-structural protein (NSP) coding region of HP-PRRSV is closely related to the high mortality rate and the structural protein (SP) coding region contributes to the induction of broadly protective neutralizing antibodies. We created a chimeric PRRSV, of which the SP coding region was derived from HP-PRRSV and NSP coding region was derived from a low-pathogenic strain. This chimeric PRRSV caused similar CPE in cells as parental viruses, but had slower growth kinetics. We hypothesize that this chimeric virus will have a low pathogenicity and could serve as a candidate vaccine that can provide protection against HP-PRRV. The second vaccine vector is a modified Listeria innocua (L.inn), a non-pathogenic strain of Listeria. Genetically related Listeria monocytogenes (L.m) is a well-known intracellular pathogen that encodes specialized virulent determinants facilitating its intracellular growth and spread. Our goal is to make L.inn a vaccine vector that can deliver classical swine fever (CSF) viral antigen into intracellular environments by complementation of L.inn with selected L.m virulence genes necessary for intracellular survival and induction of a robust immune response. In this study, we constructed a shuttle vector pHT-E2 that can express CSFV antigen E2 in L.inn. We cloned the plcA-prfA operon of L.m virulence gene cluster (vgc) into pHT-E2, which enhanced the expression of E2 in L.inn. In future studies, we plan to clone additional L.m virulence genes into the shuttle vector to increase immunogenicity of this recombinant L.inn and test its ability to protect pigs from CSFV.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) are common inhabitants of the human gastro-intestinal tract (GIT). Some LAB, especially lactobacilli, are well known for their application in fermented foods and probiotic properties. These microorganisms exert many beneficial effects on human health, such as digestion and assimilation of food and preventing pathogens colonising the GIT. Furthermore, some selected probiotic strains are believed to perform a critical role in the treatment of gastro-intestinal disorders, lactose intolerance and in the stimulation of the immune system. Despite the ever increasing consumer interest in probiotic LAB, the mechanisms by which they exert their beneficial effects and the activities of probiotics in the GIT often remain poorly understood. Understanding survival mechanisms of LAB in the GIT, especially the interaction between LAB and pathogens, would be facilitated by the direct in vivo monitoring of these processes. Using the mCherry fluorescence gene, we successfully constructed Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA reporter strains. With this study we showed that fluorescence imaging can be used to detect Lb. plantarum 423 and Ent. mundtii ST4SA in the GIT of mice. The two species colonized the cecum and colon for at least 24 h after one oral administration. To our knowledge, this is the first report on fluorescence imaging of LAB expressing mCherry in a mouse model. Using a bioluminescence marker system, we evaluated the impact of Lb. plantarum 423 and Ent. mundtii ST4SA on orally acquired Listeria monocytogenes infection and the ability of the probiotics to compete with the pathogen in the GIT of mice. Challenging Lb. plantarum 423 and Ent. mundtii ST4SA that were already established in the GIT of mice with L. monocytogenes EGDe had no effect on the survival and persistence of the probiotic strains. We demonstrated that the colonization of mice with Lb. plantarum 423 and Ent. mundtii ST4SA, or a combination of the strains, protected the animals against colonization of the GIT by L. monocytogenes EGDe. Enterococcus mundtii proved more effective than Lb. plantarum 423 in reducing the number of L. monocytogenes EGDe in the mouse model. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) kom algemeen in die mens se spysverteringkanaal (SVK) voor. Verskeie MSB, veral lactobacilli, is bekend vir hul gebruik in gefermenteerde voedsel en as probiotika. Die bakterieë het baie eienskappe wat die mens se gesondheid kan bevoordeel, insluitend vertering en assimilasie van voedsel en voorkoming van kolonisering van die SVK deur patogeniese bakterieë. Sekere probiotiese rasse speel ook ʼn belangrike rol in die behandeling van SVK versteurings, laktose intoleransie en die stimulering van die immuun stelsel. Alhoewel die belangstelling in probiotiese bakterieë toeneem, is daar min inligting bekend oor die meganismes wat MSB gebruik om hulle voordelige eienskappe in die SVK uit te voer. Die oorlewing van MSB in die SVK, veral die interaksies tussen MSB en patogene, kan met behulp van ʼn in vivo moniteringsisteem bestudeer word. Deur die mCherry fluorisensie geen in Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA te kloneer, het ons daarin geslaag om ʼn effektiewe verklikker sisteem te ontwikkel en kon die voorkoms en migrasie van die twee spesies in die SVK van muise bestudeer word. Lactobacillus plantarum 423 en Ent. mundtii ST4SA het veral die blindederm en kolon gekoloniseer. Beide rasse het na ʼn enkele mondelingse toediening vir ten minste 24 h in die SVK oorleef. Sover ons kennis strek, is hierdie die eerste verslag van fluoriserende MSB wat met behulp van die mCherry geenproduk in die SVK bestudeer is. Deur gebruik te maak van ʼn bioliggewende verklikker sisteem, het ons die vermoë van Lb. plantarum 423 en Ent. mundtii ST4SA om met Listeria monocytogenes in die SVK te kompeteer, bestudeer. Listeria monocytogenes het geen invloed gehad op die kolonisering van Lb. plantarum 423 en Ent. mundtii ST4SA nie. Deur die muise vooraf met Lb. plantarum 423 en Ent. mundtii ST4SA te koloniseer (in kombinasie of met net een van die twee rasse), kon ons daarin slaag om kolonisering van die SVK met L. monocytogenes te voorkom. In die muis model wat in hierdie studie gebruik is, was Ent. mundtii ST4SA meer effektief as Lb. plantarum 423 in die verlaging van Listeria selgetalle.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

β-lactoglobulin (β-Lg), bovine serum albumin (BSA), α-lactalbumin (α-Lac), and β-casein were adsorbed onto silanized silica surfaces of low and high hydrophobicity for 8 h, and β-Lg and BSA for 1 h. The surfaces were incubated in buffer for 0, 5, 10, or 15 h and then contacted with Listeria monocytogenes for 3 h. Cell adhesion was quantified using image analysis. Following 8 h of protein contact, adhesion to both surfaces was greatest when β-Lg was present and lowest when BSA was present. Preadsorption of α-Lac and β-casein showed an intermediate effect on cell adhesion. Adsorption of β-Lg for 1 h resulted in lower numbers of cells adhered as compared to the 8 h adsorption time, while the opposite was observed with BSA, but adhesion to BSA was observed to decrease slowly with film age to values comparable to the 8 h tests. The adsorption of BSA and β-Lg to both surfaces was also carried out where each protein was allowed to contact the surface in sequence and simultaneously. In sequential tests performed with an 8 h contact/protein, cell numbers on each surface were near that expected for the bare hydrophobic surface when β-Lg contact preceded introduction of BSA, whereas adhesion was reduced to values below that expected for the bare hydrophilic surface when BSA preceded β-Lg contact. In short-term sequential tests (1 h contact/protein), adhesion was lower than that recorded on bare hydrophilic surfaces in each case. Adhesion to each surface following contact with an equimolar mixture of β-Lg and BSA was lower than that measured on the bare hydrophilic surface in each case, with adhesion following 1 h contact being greater than that following 8 h contact. Adhesion following competitive adsorption was greater to hydrophobic than to hydrophilic surfaces. These results were explained with reference to the surface passivating character of BSA, and its ability to rapidly attain a nonexchangeable state upon adsorption, relative to β-Lg. / Graduation date: 1995

Memoria para optar al título de Ingeniera en Alimentos / Actualmente existe una gran oferta de productos desinfectantes que han enfocado su uso en la industria alimentaria para llevar a cabo de forma adecuada, programas de limpieza y desinfección, con el fin de reducir y en ciertos casos eliminar los microorganismos de la infraestructura que está en contacto con la línea de producción alimentaria. De esta forma se busca inhibir el peligro de contaminación y se garantiza un producto inocuo. Sin embargo la mayoría de los estudios con desinfectantes se realizan in vitro, no considerando los factores ambientales reales de contaminación. Es por esto que se realizó un análisis directamente en superficies ampliamente utilizadas en la industria como es el caso de acero inoxidable y plancha de polietileno de alta densidad (PEHD, por sus siglas en inglés) probando la acción bactericida de dos productos desinfectantes comercializados actualmente. El producto A compuesto por glutaraldehido y amonios cuaternarios y el producto B por ácido peracético, peróxido de hidrógeno y ácido acético. Las pruebas se realizaron a distintos tiempos y en las concentraciones indicadas por el fabricante. Para cada producto se determinó la concentración mínima inhibitoria, concentración mínima bactericida, la cinética de muerte de los microorganismos, porcentaje de eficiencia, constante específica de muerte (k), tiempo de reducción decimal (TRD) y el coeficiente de dilución. Estos análisis se realizaron frente a dos microorganismos, un Gram positivo y un Gram negativo como lo son Listeria innocua y Escherichia coli respectivamente. Los resultados obtenidos mostraron que el desinfectante A fue más eficiente que el desinfectante B, en la máxima concentración recomendada por el fabricante (0,5%) a los 5 min, logrando la disminución de al menos 6 ciclos logarítmicos, mientras que el producto B a una concentración de 0,8% logró disminuir entre 4 y 6 ciclos. Lo mismo ocurrió con las concentraciones mínimas recomendadas, donde el producto A al 0,25% logró bajar de 2 a 4 ciclos mientras que el desinfectante B solo disminuyó la carga bacteriana entre 1 y 3 ciclos logarítmicos. Fue posible determinar también a través de los valores de k y TRD, que Listeria innocua fue menos resistente que Escherichia coli frente a ambos productos evaluados, mientras que al comparar entre superficies no se encontraron diferencias entre la acción germicida sobre acero inoxidable y plancha de PEHD / Currently exists a big range of disinfectants products which has been focused its use in the Food Industry, in order to carry on cleaning and disinfection programs, with the aim to reduce and in certain cases eliminate microorganisms of the surfaces and all infrastructures what is in contact with the food production line. Thereby is sought to inhibit the contamination hazard and ensure a food safety. However, most of the disinfectants studies are done in vitro, without considering the environmental factors. Is that why it was done analysis directly performed on the most common and widely used surfaces in the Food Industry such as stainless steel and polyethylene high-density (PEHD), testing the bactericidal action of two currently marketed disinfectants. The product A, composed by glutaraldehyde and quaternary ammonium compounds as well as the product B, composed by peracetic acid, hydrogen peroxide and acetic acid. The tests were carried out at different times and at the concentrations indicated by the manufacturer. It was possible to determine the minimum inhibitory concentration, minimum bactericidal concentration, the kinetic death of microorganisms, efficiency percentage, the specific constant of death k, decimal reduction time (DRT) and diffusion coefficient. These analyses were done with two microorganisms, one Gram-positive and one Gram-negative as are Listeria innocua and Esherichia coli, respectively. The results obtained showed disinfectant A was more efficient than disinfectant B, with the maximum concentration recommended by the manufacturer (0,5%) at 5 min, achieving the reduction of at least 6 log cycles, while the product B at a concentration of 0,8% was possible to reduce between 4 and 6 log cycles. The same occurred with minimum indicated concentrations, since A at 0,25% reduced between two to four log cycles, while the disinfectant B just reduced the microbial load between 1 to 3 log cycles. It was possible to determine as well, through the k and DRT values that Listeria innocua was less resistant than Escherichia coli against both disinfectants, whereas by comparing the bactericidal action between both surfaces, no differences were found between PEHD and stainless steel surfaces

Due to renewed interest in specialty cheeses, small-scale artisan and farmstead producers are manufacturing numerous varieties of cheese, including those that present higher risk such as surface-ripened soft cheeses, often using raw milk. The presence of pathogenic bacteria in raw milk on large scale dairy farms is well documented as the dairy farm itself can serve as a reservoir. To assess the risks associated with the use of raw milk in the manufacture of small-scale artisan cheese we evaluated overall milk quality and prevalence of four target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk from 11 farmstead cheese operations in Vermont. Although overall incidence was low in comparison to other surveys, variation from farm to farm, independent of species, indicates that some operations practice strict hygienic controls and that additional effort is needed on others. Our data also suggest that if present, pathogen population levels in raw milk are extremely low. Although these pathogens are readily inactivated by pasteurization, pasteurized milk and milk products, including soft cheese, have been implicated in major outbreaks of L. monocytogenes infection as the result of post-processing environmental contamination. U.S. Standards of Identity permit the manufacture of these and other cheeses from raw milk, provided they are aged for a minimum of 60 days typically at a temperature no less than 35°F (1.67°C) to provide safety. Of particular concern are the surface-mold-ripened soft cheeses due to the growth potential during aging and refrigerated holding following increases in pH. In a study on the growth potential of L. monocytogenes introduced as post process contaminants on surface-mold-ripened cheeses we demonstrate that holding cheese in compliance with U.S. Federal regulations supports pathogen growth from very low levels regardless of the whether the milk used was raw or pasteurized. Moreover, the 60 day aging rule encourages extended holding which could inadvertently contribute to risk. It is clear that the safety of cheeses within this category must be achieved through control strategies other than pasteurization or aging. Effective environmental monitoring and control of Listeria spp. within processing plants, including farmstead cheese operations, is considered paramount in reducing cross contamination of ready-to-eat foods. To assess the incidence and ecology of Listeria spp. in farmstead cheese processing environments we conducted environmental sampling in 9 different cheesemaking facilities over a 10-week period while comparing three detection/isolation protocols. Results indicate that the use of detection/isolation methods incorporating dual primary enrichment with a repair step allowing for the recovery of injured Listeria enhances detection of Listeria spp., including L. monocytogenes, and that the addition of PCR increases sensitivity of detection while greatly reducing time to results. Our data indicate that the extent of farmstead cheese plant contamination with Listeria spp., notably L. monocytogenes, is comparatively low for dairy processing plants, especially those with contiguous farms and aids in the identification of control points for use in designing more effective control and monitoring programs. Overall the studies contained herein fill data gaps in the literature considering the threat of emerging pathogens in raw milk intended for farmstead cheesemaking, as well as the incidence and distribution of Listeria spp., including molecular subtypes, in small farmstead cheese processing environments over time. These data will help inform risk assessments which evaluate the microbiological safety of artisan cheeses, particularly those manufactured on farms.