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Various control methods used in the meat and poultry processing environment to mitigate listeriosis were evaluated using a dynamic in-plant Monte Carlo model. These control methods included food contact surface testing, sanitation, post-processing lethality treatment, and product formulation with microbial growth inhibitors. The dynamic in-plant model served as an input into the risk assessment model developed by the FDA and FSIS in 2003 which predicts the number of deaths and illnesses resulting from the use of each control method. The use of growth inhibitors combined with a post-processing lethality step was estimated to save over 200 more lives than the FSIS proposed minimum sampling standard. An analysis of data collected by the National Alliance for Food Safety and Security (NAFSS) found that retail-sliced deli meats have a greater prevalence and concentration of L. monocytogenes than prepackaged deli meats. Cross contamination at the retail level is suspected due to clustering of sample positives by store and the influence of sampling time of day on the prevalence of L. monocytogenes. The comparative risk of Listeria monocytogenes in retail sliced versus prepackaged deli meats was evaluated using a modified version of the 2003 FDA-FSIS risk assessment model which considered slicing location and the use of growth inhibitors. The comparative risk ratio for the number of deaths from retail-sliced versus prepackaged deli meats was found to be 9.1 and retail-sliced product with a growth inhibitor was found to be at greater risk for listeriosis than prepackaged product without growth inhibitor. / Master of Science

Log phase cells of Listeria monocytogenes Scott A were heat shocked in Trypticase Soy + 0.6% Yeast Extract broth at 40, 44, and 45°C for 3, 10 and 20 min at each temperature, followed by heating at 55â C for 50 minutes in order to determine an optimum heat shock response. Most heat shock temperatures significantly increased thermal resistance (p < 0.05). Increasing heat shock temperature and time allowed the organism to survive much longer at 50 to 65°C than nonheat shocked cells. The optimal heat shock condition was 45°C for 20 min where D-values at 55°C increased 2.3 fold in non-selective agar and 1.6 fold in selective agar in comparison to non-heat shocked cells. However, cells heat shocked at 48°C for 10 min gave more consistent results; these cells were heated at 50, 55, 60, and 65°C to determine a z-value. Although D-values notably increased due to heat shocking, z-values remained constant regardless of the plating medium. When aerobically heat shocked cells (45°C for 10 min) were plated on a non-selective or a selective medium, a 1.4x increase in D-value was observed when enumerated under strictly anaerobic conditions. Aerobically heat shocked cells (48°C for 10 min) added to shrimp samples retained the increased heat resistance at 55°C when enumerated on a nonselective medium compared to the non-heat shocked cells. Heat shocking conditions may be created in pasteurization or minimal thermal processing of foods allowing increased heat resistance of pathogenic and spoilage microorganisms. / Master of Science

Survival of Listeria monocytogenes in apple, orange, red grape, and white grape juice was evaluated. A six-strain cocktail of L. monocytogenes was used to inoculate (approx. 7 log cfu/ml) fruit juices, which were stored at 4, 10 and 24°C for up to 61 days. Inoculated red grape juice was stored for up to 5 hours only. Samples were withdrawn at appropriate intervals, neutralized with 1.0 N NaOH, serially diluted in 0.1% peptone water, and surface plated onto Tryptic Soy Agar + 0.6% Yeast Extract (TSAYE) and Modified Oxford Agar (MOX), followed by incubation at 32°C for 48 hours. When L. monocytogenes was no longer detected by direct plating, samples were enriched for L. monocytogenes using Listeria Enrichment Broth (LEB), followed by isolation on MOX. L. monocytogenes remained viable in white grape, apple, and orange juices for up to 12, 24 and 61 days, respectively. Over time, recovery of Listeria on TSAYE versus MOX was not significantly different (P>0.05), indicating that limited acid-injury developed during storage. The results of this study demonstrate the ability of L. monocytogenes to survive in apple, orange, and white grape juices during refrigerated and abusive storage conditions. Therefore, measures to prevent or eliminate L. monocytogenes in the fruit juice-processing environment are necessary to ensure the safety of juice products for public consumption. / Master of Science

Batches of fromage blanc, a soft white cheese were prepared from whole pasteurized cow's milk. Processing and formulation methods were used in cheese making to reduce Listeria monocytogenes in artificially contaminated cheese. Treatments implemented included use of additional starter culture in formulation (25% more starter culture than original formulation), use of a higher temperature draining process (at 45oC instead of 22oC), addition of the anti-listerial bacteriocin nisin (Danisco Nisaplin) in formulation at different levels (125 ppm, 250 ppm, 400 ppm), and combinations of these treatments. Characteristics including pH, fat content, protein content, and color were evaluated for each treatment cheese. Statistically significant differences (p<0.0001) were found between the population (log CFU/g) values of L. monocytogenes in the different treatment cheeses and control cheese. Treatments using additional starter culture or higher temperature draining alone were not successful in significantly reducing numbers of L. monocytogenes, but when combined, a 1 log reduction resulted. Of the different concentrations of nisin used in cheese formulation, the level of 250 ppm nisin was used in combination treatments. The treatments using 250 ppm nisin were able to reduce numbers of L. monocytogenes by 2 log 24h after addition. Combination treatments with 250 ppm nisin and additional starter culture in formulation reduced the level of L. monocytogenes by only 1 log, while combination treatments coupling 250 ppm nisin with a higher temperature draining and treatments with 250 ppm nisin, additional starter culture, and a higher temperature draining were able to reduce the pathogen by 2 log. There were statistically significant (p<0.0001) differences found between cheese treatments for values of pH, fat content, and protein content. This soft cheese could be standardized for each of these parameters by the processor before packaging and sale of cheese. There were no statistically significant (p>0.05) differences found between colorimetric values for different cheese treatments. / Master of Science

Tree fruit growers need information on pathogen dynamics following harvest bags contamination to determine effective sanitation interventions for decontaminating these surfaces. Therefore, the objectives of this research were (i) to determine the survival of generic E. coli, Salmonella, and L. monocytogenes on different harvest bag materials (ii) to quantify the transfer of generic E. coli, Salmonella, and L. monocytogenes from different harvest bag materials to fresh unwaxed apples and (iii) to determine the efficacy of different sanitizers for decontaminating different harvest bag materials. For Obj. 1, harvest bag materials were inoculated with rifampicin-resistant (80ppm; R) E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail. All surfaces were air-dried and held at 22 °C and either 30 or 80% relative humidity for 90 d (E. coli), or at 22 °C and 55% relative humidity (RH) for 21 d (L. monocytogenes and Salmonella). For Obj. 2, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail and air dried as previously mentioned. For E. coli trials, bacterial transfer to unwaxed 'Red Delicious' apples was assessed for 2 inoculum dry times (1 or 4 h), 2 contact times (5 or 25 minutes), and 2 pressure scenarios (0.0 or 0.1kg/cm2). For Salmonella or L. monocytogenes trials, transfer was assessed for 1 inoculum dry time (1 h), and 1 contact time (5 minutes). For Obj. 3, coupons were inoculated with L. monocytogenes or Salmonella cocktails and were air-dried. Following inoculation, coupons were exposed to different sanitizer treatments: chlorine, peroxyacetic acid (PAA), isopropyl alcohol with quaternary ammonium compounds (IPAQuats), steam, and water. Regression models were fitted, and Tukey's post hoc test was performed at P<0.05. E. coli exhibited survival for extended durations at 30 % than at 80% RH. In addition, E. coli survived at higher concentrations on canvas surfaces than on cordura and nylon surfaces. Generally, E. coli survived for more than 21 d across all surfaces and exhibited a triphasic die-off pattern. Similarly, L. monocytogenes and Salmonella exhibited die-off in phases with an initial rapid die-off followed by more gradual die-off rates up to 21 d. Canvas materials also promoted better L. monocytogenes and Salmonella survival than cordura surfaces. Contact time did not significantly impact the transfer of E. coli from harvest bag surfaces to apples (P=0.55). However, pressure, inoculum dry time and material type significantly impacted the transfer of E. coli to 'Red Delicious' apples (P≤0.03). The transfer rates of Salmonella did not differ between canvas and cordura surfaces (P=0.46). However, cordura transferred L. monocytogenes at significantly higher rates than canvas surfaces (P<0.001). Of the sanitizer treatments that were used on L. monocytogenes or Salmonella inoculated surfaces, IPAQuats was the most effective achieving over 4.5 log CFU/coupon reduction on both canvas and cordura surfaces. Our studies demonstrated that bacteria could survive for over 21 d under different conditions and could transfer from contaminated harvest bag surfaces to apples underlining the importance of cleaning and sanitizing harvest bags with sanitizers like IPAQuats. / Doctor of Philosophy / Tree fruit growers need information on pathogen behavior on harvest bag surfaces to determine effective sanitation interventions for decontaminating these surfaces. Therefore, the objectives of this research were (i) to determine the survival of generic E.coli, Salmonella, and L. monocytogenes on different harvest bag materials (ii) to quantify the transfer of generic E. coli, Salmonella, and L. monocytogenes from different harvest bag materials to fresh unwaxed apples and (iii) to determine the efficacy of different sanitizers for decontaminating different harvest bag materials. For Obj. 1, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail. All surfaces were air-dried and held for 90 d (E. coli), or 21 d (L. monocytogenes and Salmonella). For Obj. 2, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail and air dried as previously mentioned. For E. coli trials, bacterial transfer to unwaxed 'Red Delicious' apples was assessed for 2 drying conditions (wet or visibly dry), 2 contact times (5 and 25 minutes), and 2 pressure scenarios (0.0 and 0.1kg/cm2). For Salmonella or L. monocytogenes trials, transfer was assessed for wet surfaces only and apples sat on surfaces for 5 minutes. For Obj. 3, harvest bags were inoculated with L. monocytogenes or Salmonella cocktails and exposed to different sanitizer treatments including chlorine, peroxyacetic acid (PAA), isopropyl alcohol with quaternary ammonium compounds (IPAQuats), steam, and water. Regression models were fitted, and Tukey's post hoc test was performed at P<0.05. Canvas surfaces promoted better E. coli survival compared to cordura and nylon surfaces. Similarly, canvas materials also supported better L. monocytogenes and Salmonella survival compared to cordura surfaces. Contact time did not significantly impact the transfer of E. coli from harvest bag surfaces to apples (P=0.55). However, pressure, inoculum dry time and material type significantly impacted the transfer of E. coli to 'Red Delicious' apples (P≤0.03). The transfer rates of Salmonella did not differ between canvas and cordura surfaces (P=0.46). However, cordura transferred L. monocytogenes at significantly higher rates than canvas surfaces (P<0.001). Of the sanitizer treatments that were used on L. monocytogenes or Salmonella inoculated surfaces, IPAQuats was the most effective achieving over 4.5 log CFU/coupon reduction on both canvas and cordura surfaces. Our studies demonstrated that bacteria could survive for over 21 d under different conditions and could transfer from contaminated harvest bag surfaces to apples underlining the importance of cleaning and sanitizing harvest bags with sanitizers like IPAQuats.

The primary aim of this investigation was to identify and assess the interactions (synergies and antagonisms) that exist between 20 minute detergent and 5 minute disinfectant treatments upon three factory isolated strains of surface adhered (1-hour attached) and surface adapted (24-hour biofilm) populations of Escherichia coli and Listeria monocytogenes, plus a comparison with vero-toxin producing strains of E. coli, when used as part of a cleaning and disinfection regime. The detergents chosen for assessment were two non-ionic (91/4 - Alcohol Ethoxylate and KCL5 - Polyethoxylated Alcohol), two anionic (LX28 - Sodium Lauryl Sulphate and Nec28 - Sodium Laurylether Sulphate) and two novel bismuth thiols (BisEDT - 1:1 Bismuth nitrate 1,2-ethanedithiol and BisTOL - 2:1 Bismuth nitrate 3,4-dimercaptotoluene), developed at Winthrop University Hospital, New York. The disinfectants chosen for assessment were a quaternary ammonium compound (BAC - Benzyl alkonium Chloride) and a chlorine releasing agent (NaDCC - Sodium Dichloroisocyanurate). The investigation showed that there were no specific cleaning and disinfection regimes that will adequately target both E. coli and L. monocytogenes strains. It was also concluded that to maximise the removal and disinfection of persistent strains of a given microorganism, it may be necessary to design a regime to specifically target not just the species, but the strain involved and where possible requires mechanical cleaning. The novel bismuth thiols were seen to be promising detergents to aid in the removal of E. coli strains and warrant further attention for future studies. Finally, an investigation to identify possible mechanisms of resistance to disinfectant treatments following detergent treatment, showed that different detergents can induce expression of the stress response proteins, HSP60 and HSP70, at differing levels of expression after the same contact time and against different states of adherent populations, i.e. 1-hour attached or 24-hour biofilm populations.

A formação de biofilme representa preocupação à indústria de alimentos pois é uma fonte crônica de contaminação. Encontrar estratégias eficientes para controlar o crescimento de microrganismos continua a ser um importante desafio. Uma delas é o uso dos ramnolipídeos (RLs), um biossurfatante produzido tipicamente por P. aeruginosa que apresenta potencial como agente antimicrobiano, anti-adesivo e dispersivo. Sua baixa toxicidade, biodegradabilidade, eficiência e especificidade em comparação aos surfatantes sintéticos podem torná-los promissores agentes de biocontrole. O presente estudo teve como objetivo estudar o potencial de uso de ramnolipídeos, em diferentes condições de concentração e temperatura, no controle e remoção de biofilmes de patógenos alimentares formados em meio de cultura e leite. Foram utilizadas Escherichia coli ATCC 43895, Listeria monocytogenes ATCC 19112, Staphylococcus aureus ATCC 8095, reconhecidos patógenos alimentares. Os biofilmes foram formados em placas de microtitulação de poliestireno nos meios de cultivo: caldo nutriente (CN), extrato de levedura com triptona de soja (TSYE) e matriz alimentar (leite) à 37 &deg;C, por 24 h (E. coli) e 48 h (S. aureus e L. monocytogenes). Os biofilmes foram avaliados pela quantificação da biomassa, viabilidade celular, hidrofobicidade de superfície e análises qualitativa (microscopia eletrônica de varredura e de fluorescência) e quantitativa (caracterização da matriz polimérica). O ramnolipídeo foi submetido à análise físico-química de espalhamento dinâmico de luz (DLS), espalhamento de raios-X a baixo ângulo (SAXS). Os resultados obtidos para E. coli mostraram que a concentração de RL que mais removeu o biofilme foi 2 &permil;, porém em temperaturas diferentes, para o CN à 25 &deg;C e para o leite à 37 &deg;C, com 33 &permil; e 80 &permil; de remoção, respectivamente. Para o biofilme de S. aureus em caldo nutriente os resultados mais eficientes foram à 25 &deg;C, na concentração de 0,1 &permil; de RL e em leite 4 &deg;C, na concentração de 0,05 &permil; de RL, com remoção de 35 &permil; e 89 &permil;, respectivamente. O biofilme de L. monocytogenes em TSYE mostrou-se mais sensível à 37 &deg;C, na concentração 0,5 &permil; de RL, o qual foi possível remover 35,3 &permil; da biomassa. Enquanto que em leite a 4 °C e 0,5 &permil; de RL, com remoção de 63,6 &permil; .Quanto à redução das células viáveis foi observado que para as bactérias Gram-positivas o tratamento mais efetivo foi à 4 &deg;C com 0,05 &permil; de RL, nos meios CN e TSYEe 1 &permil; em leite. Para os biofilmes de E. coli a maior redução da viabilidade ocorreu em leite, após tratamento com RL 0,05 &permil; à 37 &deg;C. As imagens de microscopia mostraram uma morfologia heterogênea na presença dos diferentes meios de cultivos, com destaque para os biofilmes de S. aureus (leite) e L. monocytogenes (TSYE), nos quais houve grande produção de matriz polimérica extracelular (MPE), e também apresentaram as maiores quantidades de carboidratos e proteínas. O tratamento com o ramnolipídeo reduziu a hidrofobicidade dos biofilmes. As análises de DLS e SAXS mostraram uma predominância em número de micelas com diâmetro entre 1-10 nm, independente das concentrações e temperaturas analisadas. De modo geral, a aplicação de ramnolipídeo promoveu remoção da biomassa celular como também redução de células viáveis presentes no biofilme. As evidências obtidas aqui, podem ser importantes subsídios para futuras investigações sobre as interações físico-químicas entre ramnolipídeos e a camada de biofilme visando aplicação como agentes sanitizantes em indústria de alimentos. / Biofilm formation is a concern to the food industry because it is a chronic source of contamination. Finding effective strategies to control the growth of microorganisms remains a major challenge. One strategy is the use of rhamnolipids (RLs), a biosurfactant typically produced by P. aeruginosa that has potential as antimicrobial, anti-adhesive and biofilm disrupting agent. RLs low toxicity, biodegradability, efficiency and specificity comparatively to synthetic surfactants, makes them promising biocontrol agents. This work aimed to study the potential use of rhamnolipid at different conditions of concentration and temperature, to control and removal of biofilms of food pathogens established in culture medium and milk. The bacterial strain utilized Escherichia coli ATCC 43895, Listeria monocytogenes ATCC 19112, Staphylococcus aureus ATCC 8095, are well-recognized food pathogens. The biofilms were formed in polystyrene microtiter plates in culture media: nutrient broth (NB), yeast extract and tryptone soya (TSYE) and in food matrix (milk) at 37 &deg;C for 24 h (E. coli) and 48 h (S. aureus and L. monocytogenes). Biofilms were assessed by biomass quantification, cell viability, surface hydrophobicity, qualitative (scanning electron microscopy and fluorescence) and quantitative (characterization of polymer matrix) analysis. The rhamnolipid was subjected to physical and chemical analysis of dynamic light scattering (DLS) and X-ray small angle scattering (SAXS). E. coli biofilms were removed more efficiently using 2 &permil; RL, but at different temperatures for NB (25 °C) and milk (37 &deg;C) showing 33 &permil; and 80 &permil; respectively. For the biofilm of S. aureus in NB the best results was obtained at 25 &deg;C and 0.1 &permil; RL and in milk medium at 4 &deg;C with 0.05 &permil; RL showing 35 &permil; and 89 &permil; of biofilm disruption, respectively. The biofilm of L. monocytogenes in TSYE was more sensitive to the treatment at 37 &deg;C with 0.5 &permil; RL, removing 35.3 &permil; of the biofilm; while in milk at 4 °C and 0.5 &permil; RL, biofilm removal reached 63.6 &permil;. Reduction on cell viability was more effective for Gram-positive bacteria at 4 &deg;C with 0.05 &permil; RL, for NB and TSYE and at 1 &permil; in milk. For E. coli biofilms the largest reduction of viability occurred in milk after treatment with 0.05 &permil; RL at 37 &deg;C. The microscopy images showed a heterogeneous morphology in the presence of different media, especially biofilms of S. aureus (milk) and L. monocytogenes (TSYE), in which there was a great production of extracellular polymeric matrix (EPM), and also the highest amounts of carbohydrates and protein. The treatment with RL reduced the hydrophobicity of biofilms. The DLS and SAXS analysis of RL showed a predominance of micelles with diameters between 1-10 nm, independent of the concentrations and temperatures utilized. In general, the application of rhamnolipid promoted a reduction in biofilm mass as well in cell viability. The evidences obtained can provide a basis for future research on the physical and chemical interactions between rhamnolipid and biofilm layer aiming their application as sanitizers in food industry.

Pesquisas sobre Listeria monocytogenes em indústrias de produtos lácteos no Brasil são escassas. Três laticínios (A, B e C) produtores de queijos do Estado de São Paulo foram monitorados para a presença de L. monocytogenes no período de outubro/2008 a setembro/2009. Foram realizadas 12 coletas, correspondentes a 12 lotes de queijo produzidos, sendo quatro de cada laticínio. Em cada laticínio, as visitas foram realizadas com intervalos de aproximadamente 2 meses entre cada uma. Foram analisadas 393 amostras, sendo 201 de superfícies com e sem contato com alimentos e 192 de alimentos (leite cru e pasteurizado e queijo) água e salmoura, para pesquisa de L. monocytogenes. As análises foram realizadas de acordo com o método do Food and Drug Administration (FDA). Os resultados confirmam a presença de Listeria spp nas instalações dos três laticínios. L. monocytogenes, L. innocua, L. seeligeri e L. welshimeri foram as espécies isoladas neste estudo. Especificamente a espécie L. monocytogenes não foi encontrada no laticínio A, entretanto, o microrganismo foi isolado de 12,5% das amostras do laticínio B e de 9,1% do laticínio C. L. monocytogenes não foi isolada do leite cru dos silos, do leite pasteurizado, da água e dos queijos Minas frescal, nos 3 laticínios. Porém, no laticínio C, L. monocytogenes foi isolada de amostras de queijo Prato que foram incluídas apenas na 4ª coleta deste laticínio, além de ter sido isolada de amostras de salmoura. As maiores prevalências de contaminação por L. monocytogenes ocorreram em superfícies sem contato com alimentos, sendo positivas 51,6% das amostras do laticínio B e 21,7% do laticínio C. Em ambos os laticínios a bactéria também foi isolada de superfícies com contato com alimentos. Os resultados fornecem informações detalhadas dos pontos prioritários para o desenvolvimento de estratégias de controle de L. monocytogenes em laticínios e mostram a importância de programas de monitoramento ambiental do patógeno, mesmo em pequenas indústrias. Os 85 isolados identificados como L. monocytogenes revelaram-se de quatro sorotipos: 1/2a, 1/2b, 1/2c e 4b, com predomínio do 4b, em ambos os laticínios, o que é preocupante para a saúde pública. Com base nos resultados de PFGE (perfis combinados ApaI e AscI), 40 perfis (pulsotipos) foram obtidos. Pulsotipos foram isolados repetidamente entre coletas nos laticínios B e C, sugerindo persistência de linhagens nos laticínios. Apesar dos laticínios serem distantes e independentes, um pulsotipo foi compartilhado entre ambos. O laticínio A apresentou contaminação por mais de um pulsotipo de L. seeligeri e houve isolamento repetido de um pulsotipo dessa espécie, entre as coletas, sugerindo adaptação da bactéria e necessidade de controle do gênero Listeria nessa indústria. A ocorrência de um mesmo pulsotipo de L. monocytogenes com sorotipos diferentes (1/2b e 4b) mostra que a sorotipagem deve acompanhar análises mais refinadas como as de natureza genotípica. / Listeria monocytogenes surveys in cheese manufacturing plants in Brazil are rare. Three cheese manufacturing plants (A, B and C) in São Paulo state were monitored for the presence of Listeria monocytogenes during the period of October/2008 - September/2009. Twelve samples surveys were taken corresponding to 12 cheese lots produced, four in each plant. In each cheese plant, the samples were taken at intervals of approximately 2 months. There were 393 samples analyzed, 201 from surfaces with and without contact with food and 192 of food (raw and pasteurized milk and cheese), water and brine, with the objective of searching for L. monocytogenes. The analyses were performed in accordance with Food and Drug Administration (FDA) method. The results confirmed the presence of Listeria spp in the facilities of three plants. L. monocytogenes, L. innocua, L. seeligeri and L. weshimeri were the species isolated in this study. Specifically the L. monocytogenes specie was not isolated from plant A. However, the microorganism was isolated in 12.5% of the samples from plant B and 9.1% from plant C. Listeria monocytogenes was not isolated from raw milk in storage tanks, pasteurized milk, water or Minas frescal cheese samples from the three plants. Nevertheless, in plant C, L. monocytogenes was isolated in Prato cheese that was included only in the 4th sampling survey and also from the brine samples. The major prevalence of contamination by L. monocytogenes occurred on surfaces without contact with food, with 51.6% of the samples positive from plant B and 21.7% from plant C. In both plants, the microorganism was also isolated from food contact surfaces. The results provide detailed information about the critical points for the development of L. monocytogenes control strategies in cheese processing plants and, moreover, show the relevance of sampling programs of the pathogen, even in small cheese processing plants. The 85 isolates identified as L. monocytogenes were classified in four serotypes: 1/2a, 1/2b, 1/2c and 4b, with 4b dominating in both cheese plants, which is of concern to human health. On the basis of PFGE results (combined profiles ApaI and AscI), 40 profiles (pulsotypes) were found. Pulsotypes were isolated repeatedly among sampling surveys in plants B and C, suggesting persistence of lineages in the plants. Despite these plants being distant and independent, one pulsotype was shared between them. Plant A presented contamination by more than one pulsotype of L. seeligeri and there was a repetitive isolation of one pulsotype of this specie among samplings, suggesting adaptation of the bacterium and the need for control of the Listeria genus in this plant. The occurrence of one single pulsotype of L. monocytogenes with different serotypes (1/2b and 4b) show that serotyping should follow more refined analyses as the ones of genotypic nature.

O controle da contaminação de alimentos por Listeria monocytogenes é um desafio para a indústria, pois a bactéria é tolerante a muitas condições adversas que são empregadas para conservação de alimentos. L. monocytogenes é uma bactéria patogênica e pode causar doença de natureza não entérica grave, em pessoas imunocomprometidas, idosas e durante a gestação. No presente estudo foi avaliada a expressão gênica de duas linhagens L. monocytogenes IAL 633 sorotipo 1/2a mais comumente isoladas de alimentos e L. monocytogenes ATCC 19115 sorotipo 4b mais comumente encontradas em surtos hospitalares, utilizando caldos modelos formulados a partir de extratos de carne e peptona de peixe, suplementados ou não com glicose, cloreto de sódio, orégano, pimenta do reino e uma mistura desses ingredientes. Os caldos modelos formulados e suplementados ou não com os ingredientes foram incubados por 1 hora a 25ºC e em seguida, foi realizada extração de RNA e síntese de DNA complementar (cDNA). O cDNA foi amplificado e quantificado por PCR em tempo real, com primers para os genes alvos prfA, inlA, actA e hly, que codificam respectivamente o fator regulador positivo, internalina A, actina e hemolisina, relacionados à virulência desta bactéria. L. monocytogenes IAL 633 sorotipo 1/2a cultivada em caldo de carne acrescido de glicose diminuiu a expressão de prfA, actA e hly, enquanto que em caldo de peixe acrescido do mesmo ingrediente, foi observada maior expressão para os genes inlA, actA e hly. Nos caldos de carne acrescidos de cloreto de sódio houve aumento da expressão de hly em L. monocytogenes ATCC 19115 sorotipo 4b, mas o mesmo ingrediente causou redução da expressão dos genes inlA, actA e hly em L. monocytogenes IAL 633. Em caldo de peixe adicionado de cloreto de sódio, houve concordância no efeito observado para as duas culturas de L. monocytogenes avaliadas, com menor expressão dos genes inlA e prfA. Com pimenta do reino em caldo carne foi observado aumento da expressão de actA para L. monocytogenes ATCC 19115 e redução dos genes prfA, inlA, actA e hly para L. monocytogenes IAL 633. Já em caldo de peixe acrescido de pimenta do reino, a modulação gênica ocorreu somente para L. monocytogenes ATCC 19115, com redução do gene prfA e aumento inlA e actA. O orégano foi o ingrediente que mais afetou a expressão gênica de L. monocytogenes nas condições estudadas, com aumento da expressão de actA em caldo de carne e redução de hly, para L. monocytogenes ATCC 19115. Em contrapartida, para L. monocytogenes IAL 633 ocorreu a repressão dos mesmos genes e também de inlA e prfA, enquanto que em caldo de peixe ocorreu redução somente para o gene prfA. Ainda, em caldo de peixe acrescido de orégano foi observado redução da expressão dos genes prfA e inlA, porém aumento na expressão de actA e hly para L. monocytogenes ATC 19115. A combinação de todos os condimentos também afetou a expressão gênica, sendo que em caldo de carne houve aumento da expressão dos genes prfA e inlA, porém foi reduzida a expressão de actA para L. monocytogenes ATCC 19115. Para a mesma linhagem (ATCC 19115), em caldo de peixe ocorreu a redução da expressão dos genes prfA, inlA e actA. L. monocytogenes IAL 633 em caldo de carne com a mistura de ingredientes teve reduzida a expressão dos genes inlA e actA, essa redução também foi detectada em caldo de peixe para o gene prfA. No presente estudo foi possível observar uma repressão de todos os genes alvos estudados para L. monocytogenes IAL 633 sorotipo 1/2a quando incubada em caldo de carne suplementado com os diferentes ingredientes, mas o mesmo padrão não foi observado para L. monocytogenes ATCC 19115 sorotipo 4b, indicando a complexidade da expressão gênica em função dos componentes de alimentos. / The control of food contamination by Listeria monocytogenes is a challenge for the industry because the bacterium is tolerant to many adverse conditions applied in food preservation. L. monocytogenes can cause serious non-enteric disease in immunocompromised persons, in the elderly and during pregnancy. In the present study it was evaluated the gene expression of two strains, L. monocytogenes IAL 633 serotype 1/2a (most commonly isolated from food) and Listeria monocytogenes ATCC 19115 serotype 4b (more common in clinical cases). Model broths were prepared with meat extract and fish peptone, supplemented or not with glucose, sodium chloride, oregano, black pepper and a mixture of these ingredients. The formulated model broths supplemented or not with the ingredients were incubated for 1 hour at 25° C and next, RNA extraction was performed and complementary DNA (cDNA) was synthesized. The cDNA was amplified and quantified by Real Time PCR with primers for the target genes: positive regulatory factor (prfA), internalin A (inlA), actin (actA) and hemolysin (hly). L. monocytogenes IAL 633 serotype 1/2a grown in meat broth plus glucose decreased expression prfA, actA and hly, while in fish broth, increased expression was observed for inlA, hly, and actA genes. In meat broth added sodium chloride, increased hly expression was observed for L. monocytogenes ATCC 19115 serotype 4b but the same ingredient caused a decrease of the expression of genes inlA and hly in L. monocytogenes IAL 633. In fish broth added sodium chloride, for the two cultures of L. monocytogenes evaluated there was a lower expression of inlA and prfA gene in comparison with the control. With meat broth containing black pepper, it was observed an increased actA expression for L. monocytogenes ATCC 19115 and lower expression of prfA, inlA, actA gene; for L. monocytogenes IAL 633 lower expression of hly was observed in this broth formulation. In fish gravy plus black pepper, modulation of gene expression occurred only in L. monocytogenes ATCC 19115 that showed decrease for prfA and increase for inlA and actA. Oregano was the ingredient that affected most the gene expression of L. monocytogenes under the conditions studied, with increased actA expression in broth and hly reduction for L. monocytogenes ATCC 19115. However, for L. monocytogenes IAL 633 there was repression of the inlA and prfA genes, in fish stock was reduced only for prfA gene. Also in fish stock plus oregano was observed reduction of prfA and inlA genes, but increased expression of the actA and hly for L. monocytogenes ATC 19115. The combination of all the condiments also caused different effects on gene expression, and in broth increased the expression of genes prfA and inlA, but reduced the actA expression for L. monocytogenes ATCC 19115, for the same strain (ATCC 19115) broth fish was a reduction of prfA, inlA and actA genes. L. monocytogenes IAL 633 in broth with the mixture of ingredients reduced the expression of inlA and actA genes, this reduction were also seen in fish broth for the prfA gene. L. monocytogenes has complexity of the modulation of the expression of virulence factors in food, but in the present study it was possible to observe a repression in the modulation pattern of all target genes studied L. monocytogenes IAL 633 serotype 1/2a when incubated in broth supplemented with different ingredients, which did not occur for L. monocytogenes ATCC 19115 serotype 4b.