Challenges encountered in pathogen identification and detection include the genetic heterogeneity of strains within species of some foodborne pathogens, isolation of injured cells, mixed strains or mixed species contamination of foods, and differentiation between viable and dead cells. The first objective of this research was to evaluate an isolation medium that was based on time-delayed release (5 to 6 h) of selective agents in tablet format to a modified Listeria recovery enrichment broth (mLRB) medium for enhanced and rapid recovery of injured Listeria. The second objective involved the use of Fourier transform infrared (FT-IR) spectroscopy and chemometric analysis for the differentiation of: Listeria monocytogenes epidemic clones (ECs); viable versus heat-killed populations; different mixed strains and mixed species of Listeria; and different injury treatments and repair in Listeria populations. Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were recovered in mLRB medium, and cell populations enumerated at various times (12 to 48 h) of incubation at 37oC. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 (mLRB plus the selective agents at 6 h) were significantly higher (P < 0.05) than those in mLRBS0 (mLRB plus the selective agents at 0 h) at 24 h; however, the differences in populations on these two media were not significant for nitrite-injured Listeria. Cell populations of four strains of Listeria recovered in mLRBTD (mLRB plus the time-delayed release tablets of the selective agents) were significantly higher than when those strains were enriched in the U.S. Food and Drug Administration (FDA), International Organization for Standardization (ISO), and U.S. Department of Agriculture (USDA) broths at 24 h. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) on mLRBTD for contaminated meat than on mLRBTD for contaminated milk at 24 h. FT-IR spectroscopy in the mid-infrared region (4000 to 600 cm-1) and chemometrics was successfully applied to discriminate L. monocytogenes strains belonging to the same EC (ECII or ECIV) (100% accurate spectral classification), intact and heat-killed populations of each EC strain (100% accurate spectral classification), and spectral wavenumbers 1650 to 1390 cm-1 were used to differentiate heat-killed from intact populations. FT-IR spectroscopy and chemometrics in the wavelength region 1800 to 900 cm-1 could successfully discriminate different mixed strains of L. monocytogenes (98.15% accurate spectral classification) and different mixed species of L. monocytogenes and L. innocua (92.06% accurate spectral classification) from individual strains; Wavelength range 1800 to 900 cm-1 was successfully used to discriminate between intact, acid-injured, and heat-injured Listeria, with repaired cells from acid and heat treatments clustering closer to intact cells (93.33% of spectra accurately classified). Delayed-addition of selective agents to broth medium improves recovery of injured Listeria by allowing repair time, could minimize contamination through manual addition of selective agents, and saves analyst time; FT-IR spectroscopy is a highly discriminatory and reproducible technique that can be used for the differentiation of strains and various physiological states of Listeria.

This dissertation examined the microbial ecology of Listeria monocytogenes in three distinctly different environments: a cheese microbial community; a farm environment; and a soil microbial community. The aim of the first study was to investigate the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu cm⁻² of a L. monocytogenes six strain cocktail were conducted. The first trial had to be terminated early (day 28) as contamination of Pseudomonas spp. from the initial brine did not produce the expected characteristics of the cheese during the aging period. For the second trial, cheese samples were aged in the lab for 60 days according to the cheesemakers specifications. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms found on the surface of the rind while L. monocytogenes had little effect. In addition, although the concentration of L. monocytogenes increased to levels of 10⁶ cfu cm⁻² based on the enumeration data, the genetic data was not able to identify it in the flora due to the fact that other genera were found at much higher concentrations, which is a limitation of molecular methods used for identification of pathogens in foods. For the second study the presence and incidence of L. monocytogenes on farms that either produce raw milk cheese or supply the milk for raw milk cheese production was investigated. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested with 10 isolates found in the barn samples, 5 from environmental sites and 1 from milking equipment. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks, which emphasizes the adaptability and persistence of highly pathogenic stains in food manufacturing environments. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data and field observations we identified that drinking water sources for the animals is also an important reservoir of L. monocytogenes in farm environments. More importantly this study has shown the importance of continuous monitoring of environmental sites for the presence of the pathogen, particularly in silage. Lastly manure amended soils in the northeastern U.S. were tested for the presence and survival of rifampicin resistant Escherichia coli (rE. coli), generic E. coli (gE. coli) and Listeria spp.. Both gE.coli and rE.coli samples were processed using either direct enumeration, MPN or bag enrichment methods. Samples were taken from both tilled and surface dairy solid manure-amended plots. Listeria samples were processed using a modified BAM method. Listeria presence was constant throughout the study. In contrast, rE. coli and gE. coli levels declined with time. The main conclusions of this study were that soil type, location and physical characteristics have a significant role in the survival of bacterial populations of rE. coli, gE. coli and Listeria spp. in soil. Dairy solids application does not seem to have a long term effect on the natural microbial population of soils. Tilling of soils results in increased survival of the bacterial population due to the fact that it increases soil pore size and facilitates moisture entry, which in turn has been shown to increase bacterial survival rates. Data from this research will assist in the creation of preventative measures that lead to the elimination of pathogen reservoirs. It will be further used to verify that a 120 day interval following manure application should be sufficient to ensure food safety of edible crops subsequently planted on these soils.

Listeria monocytogenes is known to cause epidemic and sporadic cases of listeriosis. The present study investigated its occurrence, antibiotic sensitivity and serotyping of the organism in foods in various retail outlets in Gaborone, Botswana. Food samples were obtained randomly from selected supermarkets and street vendors from 5 geographical areas in Gaborone from May to September 2007. Listeria monocytogenes was isolated and positively identified by using morphological and biochemical tests. Furthermore, the organism was identified using multiplex PCR. From a total of 1324 food samples tested 57(4.3 %) were positive for Listeria monocytogenes. Out of the 57 isolates, 7 (12.3%), 3 (5.3%), 0 (0.0%), 27 (47.4%) and 20 (35.1%) were isolated from cheese, raw milk, meat (biltong), frozen cabbage and salad (coleslaw). From the 5 geographical areas selected for sampling in this study, Gaborone south recorded the most number 19 (33.3%) of L. monocytogenes isolates while Gaborone west recorded the least, 7 (12.3%). Most of the isolates (49%) belonged to serogroups 4a, 4b and 4c. These isolates were found mostly in cabbage. This was followed by serogroups 4b, 4d and 4e which comprised 30% of the isolates. This is in contrast to most studies that have found serotypes 1/2a and 1/2b to be the most common serotypes in food. That serotype 4b was detected in this study was a significant finding, because this is the number one serotype associated with human listeriosis. REP-PCR was used as a typing tool to characterize the L. monocytogenes strains. The method showed great promise as all of the L. monocytogenes strains were typable using this method, with good correlation between the REP-PCR profiles and the antibiotic resistant profiles. The findings reveal the presence of multi-drug resistant and virulent L. monocytogenes serotype 4b in ready to eat food in Gaborone, Botswana and highlight the need for education and training in food safety programmes. / Life and Consumer Sciences / M. Sc. (Microbiology (Life Sciences))

Master of Science / Department of Food Science / Elizabeth A. E. Boyle / To validate how packaging and storage reduces Listeria monocytogenes (Lm) on whole muscle turkey jerky and smoked sausage sticks, four packaging systems, including heat seal (HS), heat seal with oxygen scavenger (HSOS), nitrogen flushed with oxygen scavenger (NFOS), and vacuum (VAC), and four ambient temperature storage times were evaluated. Commercially available whole turkey jerky and pork and beef smoked sausage sticks were inoculated with Lm using a dipping or hand-massaging method, respectively. There was no interaction on packaging and storage time on Lm reduction on smoked sausage sticks and an Lm log reduction of >2.0 log CFU/cm[superscript]2 was achieved in smoked sausage sticks packaged in HS, HSOS, and VAC. A >2.0 log CFU/cm[superscript]2 reduction was achieved after 24 h of ambient temperature storage, regardless of package type. NFOS was less effective in reducing Lm by more than 0.5 log CFU/cm[superscript]2 compared to HS, HSOS or VAC. After 30 d of ambient storage, Lm had been reduced by 3.3 log CFU/cm[superscript]2 for all packaging environments. In turkey jerky, Lm reduction was affected by the interaction of packaging and storage time. HS, HSOS, NFOS, or VAC in combination with 24, 48, or 72 h ambient temperature storage achieved <1.0 log CFU/cm[superscript]2. After 30 d at ambient temperature storage, Lm was reduced by >2.0 log CFU/cm[superscript]2 in HS and VAC, and could serve as a post-lethality treatment. Alternatively, processors could package turkey jerky in HSOS or NFOS in combination with 30 d ambient storage period as an antimicrobial process. Very little data has been published describing how packaging atmospheres affects Lm survival in RTE meat. The mechanism for Lm reduction under these conditions is not fully understood and additional research is needed.

Master of Science / Food Science Institute / Kelly J. K. Getty / The United States Department of Agriculture’s Food Safety and Inspection Service require that processors of ready-to-eat (RTE) meat and poultry products implement post- processing intervention strategies for controlling Listeria monocytogenes. The objective of our study was to determine the effect of packaging methods and storage time on reducing L. monocytogenes in shelf-stable meat snacks. Commercially available kippered beef steak strips (14 × 2.5 cm rectangle piece) and turkey tenders (4 × 4 cm square piece) were dipped into a five-strain L. monocytogenes cocktail, and dried at 23°C until a water activity of approximately 0.80 was achieved. Inoculated samples were packaged with four treatments: 1) vacuum, 2) nitrogen flushed with oxygen scavenger, 3) heat sealed with oxygen scavenger, and 4) heat sealed without oxygen scavenger. Samples were stored at 23°C and evaluated for L. monocytogenes levels at 0, 24, 48, and 72 h. Initial levels (time 0) of L. monocytogenes were approximately 5.7 log CFU/cm[superscript]2 for steak and tenders. For kippered beef steak, there was no interaction among packaging treatments and storage times (P > 0.05) whereas, storage time was different (P <0.05). A 1 log reduction of L. monocytogenes was observed at 24 and 48 h at 23°C for all packaging treatments and a 2.1 log CFU/cm[superscript]2 reduction occurred at 72 h. A 1 log CFU/cm[superscript]2 reduction of L. monocytogenes was observed after 24 h of storage for turkey tenders for all packaging treatments. After 48 h of storage time turkey tenders showed >1 log CFU/cm [superscript]2 reduction of L. monocytogenes for all packaging treatments except for vacuum packaged where only 0.9 log CFU/cm[superscript]2 reduction was observed. Log reductions at 72 h for all packaging treatments for turkey tenders ranged from 1.5 to 2.2. Processors of kippered beef steak and turkey tenders could use vacuum, nitrogen-flushing, or heat sealed with an oxygen scavenger packaging methods and hold product 24 h prior to shipping to reduce potential L. monocytogenes numbers by ≥1 log. However, processors should be encouraged to hold packaged product a minimum of 72 h to enhance the margin of safety for L. monocytogenes control.

Master of Science / Food Science Institute / Elizabeth Boyle / Intrinsic factors impact Listeria monocytogenes growth in ready-to-eat poultry products. Sliced deli turkey was formulated with in-going concentrations of 1.5% NaCl or 0.75% NaCl/0.75% KCl, 0 ppm or 200 ppm NaNO[subscript]2, and using 10% or 45% pump for a total of 8 treatments. Turkey roasts were sliced and inoculated with a 5-strain L. monocytogenes cocktail or peptone water (control), vacuum packaged, and stored at 4[degree]C. Treatments were sampled on days 0, 7, 14, 21, 28, 42, 63, and 91 of storage to determine L. monocytogenes mean log growth and aerobic plate count (APC). The pH, water activity, residual nitrite concentration, and percent fat, moisture, protein, and sodium were measured using control treatments on each sampling day. There was a nitrite by day and a percent pump by day interaction (P<0.05) for L. monocytogenes and APC populations. Listeria monocytogenes populations in treatments containing 200 ppm NaNO[subscript]2 were 0.70 to 2.39 log CFU/cm[superscript]2 lower compared with products formulated with 0 ppm NaNO[subscript]2. Using 10% pump reduced L. monocytogenes populations by 0.62 to 1.50 log CFU/cm[superscript]2 on days 7 to 28 and at day 63 compared with 45% pump treatments. Incorporating 1.5% NaCl or 0.75% NaCl/0.75% KCl into formulations did not affect (P>0.05) L. monocytogenes populations during storage. On days 7 through 91, APC populations were 0.76 to 2.96 log CFU/cm[superscript]2 lower with inclusion of 200 ppm NaNO[subscript]2 compared to 0 ppm NaNO[subscript]2. There was a treatment by day interaction (P<0.05) for L. monocytogenes populations and APC. The initial inoculum level of L. monocytogenes averaged 2.21 log CFU/cm[superscript]2 and was similar (P>0.05) for all treatments on day 0. Listeria monocytogenes populations increased (P<0.05) from day 0 to 14 by 1.30 to 5.04 log CFU/cm[superscript]2. Overall, L. monocytogenes populations increased during storage and by day 91 L. monocytogenes populations were similar regardless of NaNO[subscript]2 level used except for treatments formulated with 0.75% NaCl/0.75% KCl and 10% pump. Listeria monocytogenes and APC populations were influenced by nitrite concentration and percent pump, while inclusion of NaCl or NaCl/KCl did not affect L. monocytogenes growth during refrigerated storage in vacuum packed sliced deli turkey.

Doctor of Philosophy / Food Science / Daniel Y.C. Fung / James L. Marsden / The ubiquitous nature and continued presence in food processing environments makes Listeria monocytogenes a significant threat in ready-to-eat (RTE) food products. This study was performed in two phases; Phase 1 studied lauric arginate (LAE) as an antimicrobial on food contact surfaces and shredded mozzarella cheese, and use of glucose oxidase (GOX), sodium lactate (SL), and acidified calcium sulfate (ACS) as preservatives in mozzarella cheese; Phase 2 evaluated efficacy of Photohydroionization (PHI) technology to control L. monocytogenes on food contact surfaces, sliced American cheese, and ready-to-eat turkey. Stainless steel coupons, mozzarella cheese, American cheese, and turkey were surface inoculated with a three- or five-strain cocktail of L. monocytogenes. Coupons were treated with 100 and 200 ppm solution of lauric arginate for 5 and 15 min. Mozzarella cheese was treated with different combinations of treatments comprising LAE, GOX, SL, ACS, dextrose, and anticaking agents (free flow 1031 and cellulose). Results indicated up to 2.5 log CFU/coupon reductions and it was concluded that LAE was effective in controlling low levels of contamination of L. monocytogenes on food contact surfaces. In mozzarella cheese, results indicated that lauric arginate provided no additional antimicrobial effect (P > 0.05) as compared to GOX + dextrose. The antimicrobial blends with GOX, SL, and ACS were different (P < 0.05) from the controls but showed no differences (P > 0.05) in their effect in controlling bacterial populations. Results from treatment with PHI unit showed significant (P < 0.05) reduction in bacterial populations. L. monocytogenes populations reduced by 4.37 log CFU/coupon on stainless steel surfaces after 15 min of treatment; 2.16 and 2.52 log CFU/sample reduction on American cheese and ready-to-eat turkey, respectively, after short treatment time of 5 min. Lipid oxidation analyses performed on cheese and turkey samples indicated that the PHI treatment did not affect (P > 0.05) TBAR values. These studies suggest that LAE and GOX as antimicrobials and PHI treatment can be used as intervention strategies in an integrated process to ensure safe production of food. Further research is needed to evaluate applicability of SL and ACS in mozzarella cheese.

Memoria para optar al título de Ingeniero en Alimentos / El chorizo español es un producto cárnico listo para el consumo, altamente apetecido por los consumidores de embutidos y carnes. Su intenso sabor y sus distintas formas de consumirlo lo sitúan en una posición privilegiada, sin embargo, es importante tener en cuenta los riesgos potenciales que podría tener el producto por una mala elaboración o por contaminación durante o posterior al proceso de fabricación, especialmente de tipo microbiológica, donde uno de los microorganismos más importantes tanto por su resistencia como por su nocividad es Listeria monocytogenes. El objetivo de este estudio fue determinar el efecto de la radiación sobre L. innocua como marcador de L. monocytogenes y como afecta las propiedades sensoriales en el producto. Fueron determinadas las características fisicoquímicas y microbiológicas del producto, midiendo pH, actividad de agua y recuento de contaminación natural del chorizo español con L. monocytogenes. Se determinó la sobrevida de L. innocua como marcador de L. monocytogenes en el producto inoculado durante un periodo de 11 d, almacenado en condiciones de refrigeración (4±1°C). Se realizó la dosimetría específica para el producto a través de ensayos realizados en el Irradiador experimental de 60Co, obteniendo los tiempos utilizados para cada dosis, las cuales fueron de 0,5; 1,0; 2,0 y 3,0 kGy. Se determinó el valor D10 y posteriormente se efectuó la irradiación del producto con dosis de 4D10 y 5D10 en el producto inoculado con L. innocua para ver el efecto sobre las características organolépticas del producto irradiado. El efecto en las propiedades sensoriales con las dosis utilizadas fue evaluado por un panel de 15 jueces entrenados, a través de un test triangular a los 0 y 7 d. Los valores obtenidos para pH y aw fueron 4,5 y 0,8432 respectivamente. En cuanto a la contaminación inicial de L. monocytogenes en el producto, el recuento arrojó un resultado de <10 ufc/g en todos los ensayos realizados. Para el estudio de la sobrevida de L. innocua en el producto inoculado, se obtuvo como carga inicial en el día 0 una concentración de 1,5 x 102 ufc/g, y para el día 11 una concentración de 5 x 101 ufc/g. A partir del estudio dosimétrico se obtuvieron las posiciones de máxima y mínima radiación, siendo las posiciones 2 y 5 respectivamente. También se establecieron los tiempos de irradiación para las distintas dosis utilizadas en este estudio, los cuales oscilaron entre 40 a 242 minutos aproximadamente. El valor D10 calculado fue de 0,65 kGy para el producto inoculado con L. innocua como marcador de L. monocytogenes. Los resultados del test triangular arrojaron diferencias significativas al día 0 para la muestra con mayor dosis de irradiación con respecto a la muestra no irradiada, mientras que la con menor dosis no mostró diferencias significativas. Los resultados del día 7 fueron iguales para las 2 muestras analizadas, los cuales no mostraron diferencias significativas entre las muestras, independiente de la dosis de irradiación. De acuerdo a los resultados del estudio, la radiación ionizante es un buen tratamiento para productos con alta composición grasa, pero manteniendo una dosis de irradiación menor a 5 kGy, con la cual es posible obtener una reducción de microorganismos contaminantes sin afectar la calidad sensorial / Spanish sausage is a ready-to-eat meat product highly desired by consumers of sausages and meats. Its intense flavor and its different ways of consuming it place it in a privileged position, however, it is important to take into account the potentials risks that could have the product due to poor processing or contamination during and after the manufacturing process, especially microbiological, where one of the most important microorganisms due to its resistance and harmfulness is Listeria monocytogenes. The objective of this study was to determine the effect of radiation on L. innocua as a surrogate of L. monocytogenes and how it affects the sensory properties in the product. The physicochemical and microbiological characteristics of the product were determined by measuring pH, water activity and natural contamination of the Spanish chorizo with L. monocytogenes. The survival of L. innocua as a surrogate of L. monocytogenes in the inoculated product was determined for a period of 11 days, stored under refrigeration conditions (4 ± 1° C). The specific dosimetry for the product was carried out through tests performed in a 60Co experimental irradiator, obtaining the required times for each dose, which were 0,5; 1,0; 2,0 and 3,0 kGy. The D10 value was determined and the irradiation of the product, inoculated with L. innocua, with doses of 4D10 and 5D10 was carried out in order to evaluate the effect on the organoleptic characteristics of the irradiated product. A panel of 15 trained judges, through a triangular test at 0 and 7 days, performed the sensory evaluation. The values obtained for pH and aw were 4,5 and 0,8432 respectively. As for the initial contamination of L. monocytogenes in the product, the count had a result of <10 CFU/g in all tests performed. For the study of survival of L. innocua in the inoculated product, a concentration of 1,5 x 102 CFU/g was obtained as initial loading on day 0 and for day 11 the contamination was reduced to 5 x 101 CFU/g. From the dosimetric study, the positions of maximum and minimum radiation were obtained, with positions 2 and 5 respectively. The irradiation times were also established for the different doses used in this study, which ranged from approximately 40 to 242 minutes. The calculated D10 value was 0,65 kGy for the product inoculated with L. innocua as a surrogate of L. monocytogenes. The results of the triangular test revealed significant differences at day 0 for the sample with a higher irradiation dose compared to the non-irradiated sample, while the one with the lowest dose did not show significant differences. The results of day 7 were the same for the 2 sets analyzed, which did not show significant differences between the samples, independent of irradiation dose. According to the results of the study, ionizing irradiation is a good treatment for products with high fat composition. By using an irradiation dose of less than 5 kGy, is possible to obtain a reduction of the microbiological contamination without affecting the sensory quality

O Brasil é um dos três maiores produtores mundiais de frutas, com uma produção que supera os 39 milhões de toneladas, com grande participação no mercado externo e crescente participação no mercado interno. Entretanto, não basta apenas ter quantidade, a qualidade e inocuidade do alimento são quesitos cada vez mais exigidos pelos consumidores. Tem-se conhecimento que produtos in natura podem apresentar contaminantes microbiológicos e a refrigeração é tipicamente utilizada para retardar o desenvolvimento microbiano nos produtos frescos. Microrganismos psicrotróficos, como Listeria monocytogenes, são capazes de se desenvolverem sob refrigeração. Esta bactéria é um importante patógeno causador de doenças transmitidas por alimentos e, devido a gravidade da doença e a elevada taxa de mortalidade em indivíduos suscetíveis, vem merecendo a atenção de microbiologistas de alimentos e profissionais da área da saúde. Nas frutas de baixa acidez, dentre as quais está o caqui (Diospyros kaki), L. monocytogenes pode ainda ter condições para sobreviver e se multiplicar, pois esta fruta não é submetida a tratamentos fitossanitários pós-colheita. Em muitos países, incluindo o Brasil, o caqui é consumido com a casca, logo, uma provável contaminação externa torna a situação ainda mais crítica com relação a inocuidade do produto. Assim sendo, este estudo teve como objetivos: (1) verificar a incidência de L. monocytogenes na casca de caquis (Diospyros kaki) \'Fuyu\' e \'Rama Forte\'; (2) estudar a sobrevivência e multiplicação daquele patógeno na casca e na polpa dessa fruta em diferentes tempos e temperaturas de incubação; (3) e obter parâmetros de crescimento (a duração da fase lag, o tempo de geração e a taxa de crescimento exponencial). A pesquisa de incidência de L. monocytogenes na superfície dos caquis foi realizada durante os meses de março a julho nos anos de 2005 e 2006. As amostras foram coletadas no comércio varejista na região de Campinas, interior do Estado de São Paulo, e no atacado, sendo o CEAGESP o local escolhido para esse último tipo de coleta. Um total de 582 frutos foram analisados pelo sistema Bax®, que é um método baseado na Reação em Cadeia da Polimerase (PCR). A habilidade desse patógeno se desenvolver nas duas variedades de caqui estudadas também foi verificada na polpa e na casca das frutas em diferentes tempos de incubação e a temperaturas de 10ºC, 20ºC e 30ºC. Os parâmetros de crescimento de L. monocytogenes (duração da fase lag, taxa de crescimento exponencial e tempo de geração) foram obtidos a partir dos dados experimentais. A pesquisa de incidência revelou ausência de L. monocytogenes nas cascas das frutas. Os dados de crescimento obtidos para o patógeno indicam que este pode sobreviver e se multiplicar tanto na polpa quanto na casca dos caquis \'Fuyu\' e \'Rama Forte\', e que a baixa temperatura pode retardar o seu crescimento, mas não evitá-lo. / Brazil is among of the three biggest fruit producer in the world, with a production that is over 39 million tons, a relevant participation in the international market and an increasing participation in the domestic market. However, beyond quantity the quality and safety of food are issues equally demanded by consumers. It has been known that in natura products can contain microbiological contaminants and cooling is normally used to retard microbial development in fresh produce. Psychotrophic microorganisms, such as Listeria monocytogenes, are able to develop under refrigeration. This bacterium has been recognized as an important foodborne pathogen and, due to the severity of the disease and the high mortality in susceptible individuals, it has been deserving the attention of food microbiologists and health professionals. In fruits of low acidity, in which persimmon fruit is included, L. monocytogenes can have conditions to survive and to multiply, because it is not submitted to post harvest treatments. In many countries, including Brazil, persimmon is consumed with its skin, so an external contamination may make the situation even more critical with respect to the product safety. So the objectives of the present study were: (1) to verify the incidence of L. monocytogenes on the skin of \'Fuyu\' and \'Rama Forte\' persimmons (Diospyros kaki) fruits; (2) to study the survival and multiplication of that pathogen on the skin and in the pulp of this fruit at different times and temperatures of incubation; (3) to obtain kinetic values (lag phase duration, generation time and exponential growth). The incidence of L. monocytogenes on the skin of persimmon was verified from March to July in the years of 2005 and 2006. Samples were collected from retail in the city of Campinas, State of Sao Paulo, and from wholesale CEAGESP. A total of 582 fruits were analyzed using the Bax® System, which is a method based on Polymerase Chain Reaction (PCR). The ability of this pathogen to develop in both varieties of persimmons studied was also verified on the surface and in the pulp of the fruits at different times of incubation and at 10ºC, 20ºC and 30ºC. Kinetic values of L. monocytogenes (lag phase duration, generation time and exponential growth) were obtained from experimental data. The incidence survey showed the absence of L. monocytogenes on the skin of the fruit. The growth data showed that this pathogen can grow on the surface as well in the pulp of the \'Fuyu\' and \'Rama Forte\' persimmon fruits and that low temperatures can reduce the generation rate but does not inhibit its growth.

Listeria monocytogenes (L. monocytogenes) est une bactérie ubiquitaire Gram-positive aux niches écologiques et porteurs divers. L'infection humaine, par ce micro-organisme, liée à la consommation d’aliments contaminés peut provoquer une infection rare, mais grave, la listériose. L'identification et le typage de cette bactérie sont des étapes importantes pour la maîtrise des dangers à chaque étape de la chaîne alimentaire. Les méthodes de typage disponibles que sont le sérotypage, le sérotypage moléculaire, la PFGE (Electrophorèse en Champs pulsé) et la MLST (Multiloci Type de séquence), ont permis de comprendre la diversité de L. monocytogenes et d’endiguer des épidémies. Ces dernières années, les NGS (Next Generation Sequencing) et le développement de méthodes de calcul ont rendu possible l'application du séquençage du génome entier (WGS) comme outils de typage. Dans cette étude, nous avons profité de la vaste collection de souches de L. monocytogenes isolées de l'alimentation et gérée par l'ANSES. Les outils de typage standards comme les protocoles WGS ont été appliqués et comparés sur cette collection. Nous avons observé que la population de L. monocytogenes est très structurée, quel que soit l’outil de typage utilisé, de plus, les résultats sont concordants. Chaque groupe de souches, désigné comme "séquence type " (ST) ou "complexe clonal" (CC) dans ce travail, montre des caractéristiques spécifiques sur leur fréquence, les résultats de typage, l'association à l'aliment ou au cas clinique et le profil de virulence. Ces données permettent de classer plus finement les souches de L. monocytogenes et de prédire le potentiel pathogénique de souches. De plus, les WGS permettent la surveillance en routine, la détection d'épidémies, l’identification des sources de contamination et des risques, de par leur forte résolution. Avec le programme « BlastP », une base de données de gènes associés à la virulence et le panel de souches de l'ANSES, nous avons pu distinguer un gène de virulence, InlF (internalin F), lequel, tronqué, pourrait expliquer la fréquence extrêmement faible de cas cliniques dans le groupe de souches ST121.Les WGS représentent une nouvelle étape vers une meilleure appréciation et gestion des risques de santé liés à L. monocytogenes. Cependant, des améliorations comme une nomenclature commune, des protocoles standardisés pour les WGS et des outils simplifiés pour partager des données et ainsi renforcer l'efficacité de la surveillance de L. monocytogenes seraient nécessaires / Listeria monocytogenes (L. monocytogenes) is a gram-positive bacterium present in diverse ecological environments and hosts. The microorganism may infect humans and these infections can often be traced back to contaminated foods. The bacterium can sometimes lead to rare but serious infection and in particular a lethal infection known as listeriosis. The bacterium can enter the food chain at all production stages and therefore identification and characterisation of this bacterium are critical steps in the control of potential hazards to the food chain. The current available characterisation methods, which include serotyping, molecular serotyping, PFGE (Pulsed-Field Gel Electrophoresis) and MLST (Multilocus Sequence Type), have provided understanding about the diversity of L. monocytogenes. Further the characterisation methods can facilitate the investigation of outbreaks. During the last few years, (NGS) Next Generation Sequencing and development of computational method for genome wide studies have made it possible to apply WGS (whole genome sequencing) as a typing tool. In this study, we took advantage of the large and the well-characterized collection of L. monocytogenes strains isolated from foods, which is available at Anses. Standard typing tools as well as recent WGS protocols were applied and compared. We observed that L. monocytogenes population could be distinctly separated and structured when analysed by diverse typing tools. Moreover, the investigation displayed consistency among the typing tools. Each cluster of strains, commonly referred to as Sequence Type (ST) or Clonal Complex (CC), shows specific features regarding prevalence, typing results, association to food or the clinical and virulence profile. It opens the possibility for a detailed classification of L. monocytogenes and the possibility to predict the potential pathogenicity of strains based on knowledge of those specific features. By utilising the BlastP program, a virulence associated genes database and the panel of strains housed by Anses, we identified one gene, internalin F (InlF), truncated specifically in ST121. It could therefore explain the observed low frequency of clinical case among strains from ST121.WGS represents a step towards even better identification and management of health risks related to L. Monocytogenes. However, in order to enhance efficiency of foodborne pathogen surveys it would be necessary to implement improvements such as common nomenclature, standard WGS protocols and uniform standards for data sharing