Listeria monocytogenes é de grande preocupação para a sande pública e para indústrias de alimentos, pois pode causar meningite, septicemia e aborto. Trabalhos desenvolvidos em diversos países, bem como no Brasil, indicam que a presenca de L. monocytogenes nas indústrias processadoras de pescado é freqüente. Corn a finalidade de monitorar a contaminação por L. monocytogenes em plantas processadoras de alimentos e identificar as fontes de contaminação do produto têm-se empregado técnicas de biologia molecular. Dentre elas cabe destacar a PFGE (pulsed field gel electrophoresis - eletroforese em gel de campo pulsado), técnica baseada em macrorestrição genômica, através da utilização de uma enzima de restrição de baixa freqüncia de corte, como a SmaI , AscI e ApaI, seguido de uma eletroforese em gel em campo pulsado. Neste estudo foram utilizadas 181 cepas de L. monocytogenes isoladas a partir de amostras de salmão \"gravlax\" colhidas nas diferentes etapas de processamento, além de amostras de manipuladores, ambientes e utensílios, coletadas em uma usina de processamento industrial a fim de verificar a diversidade antigênica e genética. Estas cepas foram sorogrupadas corn os antissoros 0 tipo 1 e 4 e subtipadas após PFGE seguindo o protocolo preconizado pelo Center for Disease Control and Prevention e empregando as enzimas ApaI e AscI. Das cepas, 132 (72,9%) pertenceram ao sorogrupo 1 e 49 (27,1%) ao sorogrupo 4. Corn as enzimas ApaI e AscI foram obtidos, respectivamente, 30 e 28 perfis de macrorestriçãoo diferentes. Os perfis de ambas as enzimas foram combinados obtendo-se 60 perfis combinados de macrorestrição. A combinação dos perfis de macrorestrição à sorologia permitiu separar as cepas em 8 subtipos e a distribuição destes subtipos na linha de processamento foi avaliada. Notou-se que um mesmo perfil de macrorestrição foi encontrado no salmão desde o início do processamento até o produto final, já embalado, utensílios e nas mãos de manipuladores. Também verificou-se que alguns perfis estavam adaptados ao ambiente de processamento e utensílios não sendo transmitidos ao produto. Na planta em questão há necessidade de aplicação inicialmente de Boas Práticas de Fabricação (BPF) e posteriormente Análise de Perigos e Pontos Críticos de Controle (HACCP). / Listeria monocytogenes is a great concern for the public health and for the food industry, because it can cause serious illness such as meningitis, septicemia and abortion. Lots of studies in many countries, including Brazil, point to the frequent presence of L. monocytogenes in the fish industries. Molecular biology techniques have been applied to trace the contamination of L. monocytogenes and identify the sources of contamination of the food processing lines. One of them is the PFGE (pulsed-field gel electrophoresis), which is based on a genomic macrorestriction with low frequency restriction enzymes (SmaI, AscI and Apal), followed by a pulsed electrophoresis. 181 strains of L. monocytogenes were isolated from \"gravlax\" salmon processing line in several stages of the industrial processing, handworkers, environmental and food contact surfaces samples and the antigenic and genetic diversity were evaluated. They were serogrouped with antiserum 0 type 1 and 4, and subtyped using PFGE according to Center for Disease Control and Prevention protocol using the enzymes ApaI and AscI. 132 strains (72,9%) belonged to serogroup 1, 49 (27,1%) to serogroup 4. Visual comparison for macrorestriction patterns revealed 30 distinct ApaI restriction endonuclease digestion profiles (REDP) and 28 AscI REDP. When the composite profile from both enzymes was generated, there were 60 profiles. The combined macrorestriction profiles were joined to the serology resulting in 8 subtypes and the subtypes distribution along the processing line was evaluated. It\'s interesting to note that one specific profile found in the raw material was also found in different processing steps up to the final product (readyto-eat \"gravlax\" salmon) and also in food contact and non food contac surfaces, as well as food handlers. Some profiles were well adapted to the processing environment and were not found in fish. Our results strongly suggest that this industry needs to apply the Good (GMP) and Analysis of Hazards and Critical Points of Control (HACCP) to produce safer products.

Listeria monocytogenes é conhecida como um importante patógeno causador de doenças transmitidas por alimentos na última década. Apesar do número de casos por ano ser relativamente baixo, a infecção pode ser grave, com mortalidades acima de 30%. Pesquisas realizadas no Brasil relataram uma incidência de 32% em amostras de produtos cárneos, e o microrganismo foi encontrado em 80% das amostras lingüiças frescal de carne suína. Apesar dos recentes avanços nas tecnologias de controle de patógenos em alimentos, os consumidores têm procurado alimentos \"naturais\", isto é, submetidos a tratamentos menos agressivos e isentos de conservadores químicos. Antimicrobianos naturais são uma opção interessante, mas sua aplicação requer uma melhor compreensão de sua funcionalidade nos alimentos. Os óleos essenciais e seus compostos fenólicos estão se tornando agentes antimicrobianos naturais bastante populares, assim como a nisina, uma bacteriocina produzida por Lactococcus lactis subsp. lactis. Esta pesquisa foi desenvolvida para avaliar o efeito de óleo essencial de orégano (O.E.O.) e de nisina, individualmente ou em combinação, na inibição da multiplicação de Listeria monocytogenes Scott A in vitro (meio de cultura) e in situ -(lingüiça frescal suína). A atividade inibitória foi testada pela metodologia de difusão em poços, e os halos de inibição foram medidos 24 horas após a incubação à 37ºC. Foram testadas as concentrações 0,05%, 0,1%, 0,2%, 0,3%, 0,4% e 0,5% (v/v) de O.E.O. e 0, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 e 2000 ppm de nisina. Quando o óleo essencial foi usado em combinação com a nisina foi observado um efeito sinérgico na inibição de L. monocytogenes. As concentrações que apresentaram o maior efeito contra o patógeno nos testes in vitro, ou seja 0,5% (v/v) O.E.O. com 200 ppm de nisina, foram utilizadas nos experimentos com três formulações diferentes lingüiça frescal, contendo pernil de porco, sal (2%), nitrito (0,015%), condimentos, emulsificantes e antioxidantes, experimentalmente contaminadas com L. monocytogenes Scott A (106 UFC/g). A multiplicação do patógeno foi monitorada no produto refrigerado a 5oC por até 10 dias, através da contagem em placas. Controles sem antimicrobianos também foram estudados. Os resultados indicaram que o O.E.O., usado isoladamente, não conferiu proteção ao alimento. A nisina causou uma redução de 2 log imediatamente após o contato com o microrganismo, mas durante o armazenamento, as células sobreviventes apresentaram a mesma taxa de multiplicação que na lingüiça controle (??0,05), mantendo as contagens 2 log inferiores as do controle por até 9 dias. Quando os dois antimicrobianos foram usados em combinação, a redução imediata após o contato foi de 4 log, e, quando comparado ao controle, a taxa de multiplicação durante o armazenamento a 5oC foi significativamente mais altas que no controle (??0,05). Entretanto, as amostras de lingüiça contendo esses antimicrobianos nas concentrações testadas não foram aprovadas nos testes sensoriais de aceitação (??0,05). Esses resultados indicam que a combinação desses antimicrobianos pode ser utilizada como uma barreira adicional para a multiplicação de L.monocytogenes em lingüiça frescal suína, mas os atributos sensoriais que conferem ao produto podem limitar sua aplicação. / Listeria monocytogenes has been recognized as an important foodborne pathogen for the past decade. Although the number of cases per annum is relatively low, the infections can be acute, with mortality up to 30%. In Brazil, some works reported that 32% of dairy meat products were contaminated with L. monocytogenes, and this organism was found in 80% of fresh pork sausage. In spite of modern improvements in food production techniques, the consumers are seeking for \"natural\" food products, i.e., not submitted to aggressive treatments or added of chemical preservatives. Natural antimicrobials are a promising option, but their application requires a better understanding of their functionality in foods. Naturally occurring antimicrobial agents, such as essential oils and their phenolic components, are becoming increasingly popular as preservation agents. Other compound with increased application in foods is nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis. This study aimed to evaluate the antimicrobial effect of oregano essential oil (O.E.O.) and nisin, individually or in combination, on the inhibition of growth of Listeria monocytogenes Scott A in vitro (agar culture medium) and in situ (fresh pork sausage). The inhibitory activity was tested by the well diffusion method, measuring the inhibition halos after 24hours incubation at 37ºC. The concentrations tested were 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (v/v) for OEO and 0, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 ppm for nisin. When the essential oil was used in combination with nisin, a synergistic effect was observed for L. monocytogenes, i.e., the oil enhanced the activity of the bacteriocin. The combination 0.5% (v/v) O.E.O. with 200 ppm nisin presented the best results and was used to test the functionality in fresh pork sausage prepared with three different formulations, containing deboned minced pork meat, 2% salt and 0.015% nitrite, plus spices, emulsifier and antioxidant, and experimentally contaminated with L. monocytogenes Scott A (106 CFU/g). The growth of the pathogen was monitored in the refrigerated product (5oC) up to 10 days, by means of plate counting. Controls without antimicrobials were included in the experiments. Results indicated that O.E.O., used alone, was not effective. Nisin alone caused a 2-log reduction immediately after contact, but during storage the surviving cells presented the same multiplication rate as in the control (? ? 0.5), keeping the counts 2 log lower up to 9 days. When used in combination, the two antimicrobials caused a 4-log count reduction immediately after addition and, when compared to the control, the multiplication rate of the surviving cells during storage under refrigeration up to 10 days was significantly higher (? ? 0.5). However, samples containing these antimicrobials in the tested concentrations failed the sensorial acceptance tests (? ? 0.5). These results indicate that the combination of these antimicrobials can be an additional hurdle for the control of L. monocytogenes in fresh pork sausages, but the final sensorial attributes of the product may hamper their application.

Não consta resumo na publicação. / Abstract not available.

Listeria monocytogenes causa a listeriose, uma doença zoonótica grave que causa infecções do sistema nervoso central (meningite, encefalite e meningoencefalite), bacteremia primária e septicemia. A doença apresenta baixa morbidade e alta mortalidade e acomete, principalmente, grupos de risco, como mulheres grávidas, neonatos, indivíduos imunocomprometidos e idosos. L. monocytogenes tem sido encontrada com freqüência em alimentos in natura e/ou processados, como queijos e outros produtos lácteos. Esse estudo objetivou isolar bactérias lácticas a partir de leite de cabra, capazes de produzir peptídeos antimicrobianos (bacteriocinas), identificar estas cepas, caracterizar as bacteriocinas produzidas e avaliar o seu potencial de aplicação no controle da multiplicação de L. monocytogenes em queijo de cabra durante armazenamento a 8-10°C. Trabalhando-se com leite de cabra cru, foi possível isolar seis cepas produtoras de bacteriocinas (DF2Mi, DF3Mi, DF4Mi, DF5Mi, DF6Mi e DF60Mi). Através de testes fenotípicos apropriados e sequenciamento do 16S rRNA, essas cepas foram identificadas como Lactococcus lactis subsp. lactis (DF2Mi, DF3Mi, DF4Mi e DF5Mi), Leuconostoc lactis (DF6Mi) e Lactobacillus paracasei subsp. paracasei (DF60Mi). A caracterização físico-química e biológica das bacteriocinas produzidas pelas cepas DF4Mi, DF6Mi e DF60Mi indicou que eram resistentes ao calor e extremos de pH, mas apresentavam características diferentes em relação ao espectro de ação, sensibilidade a agentes químicos, adsorção à células-alvo e lise das células de L. monocytogenes. O efeito do pH, temperatura e composição do meio de cultura na produção das bacteriocinas foi também cepa-dependente. A cepa DF4Mi apresentou melhor atividade antimicrobiana e foi selecionada para o estudo de inibição de L. monocytogenes em queijos. Para isso, foram preparados lotes de queijo frescal, feito com leite de cabra pasteurizado adicionado e não adicionado da cepa DF4Mi (106 UFC/mL), experimentalmente contaminado com L. monocytogenes (103 UFC/g), além dos controles positivo (queijo adicionado de nisina 12,5 mg/Kg) e negativo (leite adicionado de uma cepa de L. lactis subsp. lactis não bacteriocinogênica). Observou-se que a cepa DF4Mi apresentou efeito bacteriostático no queijo, sendo capaz de inibir a multiplicação do patógeno durante o armazenamento a 8-10°C por 10 dias. No entanto, inibição semelhante foi obtida nos queijos com a bactéria lática não bacteriocinogênica, indicando que a inibição não pode ser creditada às bacteriocinas. Nos queijos-controle com nisina, foi observada uma redução de 2 log na contagem de L. monocytogenes após 10 dias a 8-10°C. Já nos queijos preparados sem nisina e sem nenhuma bactéria lática, as contagens de L. monocytogenes atingiram contagens elevadas (106 UFC/g) após 10 dias de armazenamento a 8-10°C. / Listeria monocytogenes causes listeriosis, a serious zoonotic disease that causes infections in the central nervous system (meningitis, encephalitis and meningoencephalitis), bacteremia and septicemia. The disease presents low morbidity and high mortality and affects mainly those in the risk group, such as pregnant women, neonates, immunocompromised individuals and the elderly. L. monocytogenes has been frequently detected in in natura and processed foods. Like cheeses and other dairy products. This study aimed to isolate lactic acid bacteria capable of producing antimicrobial compounds from goat milk, identify the isolates, characterize the bacteriocins and evaluate their potential application in controlling the growth of L. monocytogenes in goat cheese during storage at 8-10°C. Six bacteriocinogenic strains (DF2Mi, DF3Mi, DF4Mi, DF5Mi, DF6Mi e DF60Mi) were successfully isolated from raw goat milk. Using appropriate phenotypic tests and 16S rRNA sequencing, these strains were identified as Lactococcus lactis subsp. lactis (DF2Mi, DF3Mi, DF4Mi e DF5Mi), Leuconostoc lactis (DF6Mi) and Lactobacillus paracasei subsp. paracasei (DF60Mi). The physico-chemical and biological characterization of the bacteriocins produced by the strains DF4Mi, DF6Mi e DF60Mi indicated that they were resistant to heat and pH extremes, but presented different spectrum of activity, sensitivity to chemicals, adsorption to target cells and lysis of L. monocytogenes. The effect of pH, temperature and culture media composition in bacteriocin production was also strain-dependent. The strain DF4Mi presented the best antimicrobial activity and was selected for the studies on inhibition of L. monocytogenes in cheese. Frescal cheese was manufactured with pasteurized goat milk added of a culture of DF4Mi (106 CFU/mL), and experimentally contaminated with L. monocytogenes (103 CFU/g). Control cheeses were also prepared: those added of 12.5 mg/Kg nisin (positive control) and those added of a non-bacteriocinogenic L. lactis subsp. lactis strain. The strain presented a bacteriostatic effect, controlling the growth of the pathogen for 10 days at 8-10°C. However, a similar effect was observed in the cheeses prepared with the non-bacteriocinogenic strain, indicating that the inhibition cannot be credited to bacteriocins. In the cheeses containing nisin, a 2 log reduction in the counts of L. monocytogenes was achieved after 10 days at 8-10°C. In the cheeses with no added nisin or lactic acid bacteria, the counts of L. monocytogenes after 10 days at 8-10°C were high (106 CFU/g).

O efeito combinado de bacteriocina produzida por Lacfobacillus sake 2ª e embalagem em atmosfera modificada sobre o controle de Lisferia monocytogenes F5069r em lingüiça frescal foi avaliado. A cepa L. sake 2a foi co-inoculada com L. monocytogenes F5069r (resistente a cloranfenicol e eritromicina) em lingüiça frescal. As lingüiças foram embaladas com ar, 100% CO2 ou 50%CO2/50%N2 e armazenadas a 6°C. A multiplicação de L. Monocytogenes F5069r e L. sake 2a foi monitorada durante 4 semanas em intervalos de 7 dias. A avaliação sensorial, por meio do teste triangular, foi realizada após 5 e 11 dias, os quais foram estipulados de acordo com a vida-de-prateleira do produto. Após 28 dias de estocagem, a população de L. monocytogenes nas amostras inoculadas com L. sake 2a e embaladas com atmosfera modificada foi 6.4 ciclos logarítmicos menor que no controle sem a bactéria lática e embalado em ar. No entanto, a influência da atmosfera modificada sobre as características sensoriais do produto foram detectáveis após cinco dias de estocagem, independente da adição de L. sake 2a. Ao final da primeira semana, a influência de L. sake 2a sobre L. monocytogenes foi menos importante (redução de 0,4 log, não significante) que a influência da embalagem em atmosfera modificada (redução de 1,4 log, significante). No décimo primeiro dia, nenhuma diferença sensorial foi encontrada entre as amostras com e sem L. sake 2a embaladas em atmosfera modificada. Após 14 dias, a população de L. monocyfogenes nas amostras com L. sake 2ª embaladas em atmosfera modificada foi 3,5 log menor que no controle sem a bactéria lática e embalado em ar. Os resultados sugerem que o uso combinado de atmosfera modificada e bacteriocina produzida por L. sake 2a apresenta um efeito sinergístico sobre o controle de L. monocytogenes F5069r em lingüiça frescal refrigerada. / Lactobacillus sake 2a is a bacteriocinogenic strain isolated from \"lingüiça frescal\", a Brazilian sausage. The combined effect of modified-atmosphere packaging and addition of L. sake 2a on inhibition of L. monocyfogenes in \"lingüiça\" was evaluated. Samples were inoculated with L. monocytogenes and/or L. sake 2a, packed with oxygen-permeable film, 100%CO2 or 50%CO2+50%N2 and stored at 6°C. Microbial counts were performed weekly. Sensorial evaluation (triangle tests with 16 subjects) was performed after 5 and 11 days (shelf-life). After the fourth week, L. monocytogenes population in samples packed with modified atmosphere containing L. sake 2a was 6.4 log lower than in samples without any treatment. However, the influence of the modified atmosphere on the sensorial characteristics of the product was already detectable on the fifth day (a risk of 5%), regardless the addition of L. sake 2a. By the end of the first week, the influence of L. sake 2a on the inhibition of L. Monocytogenes was less important (reduction of 0.4 log, non significant) than the influence of the packaging (reduction of 1.4 log, significant). On the 11th day, no significant sensorial difference was found between the samples with and without L. sake 2a packed with modified atmosphere. By the end of the second week, L.monocytogenes counts in samples packed with modified atmosphere containing L. sake 2a were 3.5 log lower than counts in samples without any treatment. Combination of results suggests that modified atmosphere and L. sake 2a act synergistically on inhibition of L. monocytogenes in \"lingüiça frescal\".

<p>Ispitivan je uticaj vrednosti procesnih parametara: na tehnolo&scaron;ku efikasnost procesa osmotske dehidratacije pilećeg mesa u vodenom rastvoru NaCl i saharoze i melasi; na nivo redukcije odabranih mikroorganizama (Escherichia coli, Salmonella spp., Listeria monocytogenes) u osmotskim rastvorima; na mikroorganizme prisutne na dehidrirajućem pilećem mesu; istraživana je podobnost osmotski dehidriranog pilećeg mesa za rast i razmnožavanje odabranih mikroorganizama tokom perioda skladi&scaron;tenja, uz definisanje zdravstveno bezbednog roka skladi&scaron;tenja na osnovu mikrobiolo&scaron;kih i hemijskih analiza.<br />Rezultati ispitivanja pokazuju da povećanje vrednosti procesnih parametara temperature, vremena trajanja procesa i koncentracije osmotskih rastvora dovodi do intenziviranja prenosa mase između dehidrirajućeg materijala i rastvora i povećanja efikasnosti procesa. Izlaganjem odabranih mikroorganizma osmotskim rastvorima postignuti su visoki nivoi njihove redukcije. U melasi postignuti su vi&scaron;i nivou redukcije mikroorganizama u poređenju sa vodenim rastvorom. Ostvareni nivoi redukcije odabranih mikroorganizama na pilećem mesu tokom procesa niži su u poređenju sa rezultatima redukcionih odnosa istih mikroorganizama direkno inokulisanih u istim osmotskim rastvorima. Sa protokom vremena skladi&scaron;tenja ve&scaron;tački kontaminiranog i osmotski dehidriranog pilećeg mesa, u oba osmotska rastvora, do&scaron;lo je do smanjenja broja svih ispitivanih mikroorganizama. Proteolitički mikroorganizami nisu bili prisutni u dehidranom pilećem mesu, dok sadržaj histamina je pokazao da, tokom vremena skladi&scaron;tenja, nije dolazilo do degradacije proteina u mesu. Nakon 10 dana skladi&scaron;tenja meso nije bilo užeglo, a vrednosti malondialdehida su ukazale na pojavu užegnuća nakon 14 dana skladi&scaron;tenja.<br />Na osnovu dobijenih rezultata razvijeni su modeli zavisnosti odziva procesa osmotske dehidratacije, nivoa redukcije ispitivanih mikroorganizama u osmotskim rastvorima, nivoa redukcije ispitivanih mikroorganizama na dehidriranom pilećem mesu i mikrobiolo&scaron;kih i hemijskih odziva dehidriranog pilećeg mesa tokom skladi&scaron;tenja u zavisnosti od variranih vrednosti parametara procesa.<br />Na osnovu dobijenih rezultata, kao optimalni parametri, mogu da se defini&scaron;u: trajanje procesa od 5 časova, temperatura od 32&deg;C u melasi, kao osmotskom rastvoru, maksimalne koncentracije. Svi postignuti nivoi redukcije mikroorganizama ukazuju na dobru osnovu za proizvodnju zdravstveno bezbednih proizvoda od pilećeg mesa. Analiza održivosti je pokazala da je osmotski dehidriranom pileće meso mikrobiolo&scaron;ki i hemijski stabilno tokom skladi&scaron;tenja na temperaturi od 22 &deg;C u trajanju od najmanje 10 dana.</p> / <p>The effect of process parameters values on: technological efficiency of chicken meat osmotic dehydration process in water solution of NaCl and succrose and molasses; selected microorganisms (Escherichia coli, Salmonella spp., Listeria monocytogenes) in osmotic solutions reduction levels; selected microorganisms on dehydrating chicken meat reduction levels, is investigated. The osmodehydrated chicken meat suitability for selected microorganisms&rsquo; growth and multiplication during storage period is also investigated, together with defining health safe storage period, on the basis of microbiological and chemical analysis.<br />Results shows that increase of process parameters of temperature, duration and osmotic solutions&rsquo; concentrations leads to mass transfer increase between dehydrating material and osmotic solutions and process efficiency increase. Exposure of selected microorganisms to osmotic solution has led to high levels of reductions of their numbers. Processes in molasses had higher levels of microorganisms&rsquo; reductions in comparison to the water solution. Achieved levels of the selected microorganisms&rsquo; on chicken meat reductions were lower in comparison to the results of reduction of the same microorganisms directly inoculated in the same osmotic solutions. With the increase of the inocualted, osmotically dehydrated chicken meat storage time in both osmotic solutions, decrease of all tested microorganisms occured. Proteolytic microorganisms were not detected in dehydrated chiken meat, while histamin content showed that, during storage, there was no meat protein degradation. After 10 days of storage, meat was not rancid, while malondialdehid values showed that lipid oxidation occured after 14 days of storage. On the basis of obtained results, mathematical models of dependance of: osmotic dehydration process responces; selected micororganisms in osmotic solutions reduction levels; selected microorganisms on osmodehydrated chicken meat reduction levels; and osmodehydrated chicken meat during storage microbiological and chemical responces; from varied process parameters, were developed.<br />Based on obtained results, as optimal process parameters it can be defined: 5-hour process, at 32 &deg;C, in molasses of maximal concentration, as osmotic solution. All achived microorganisms&rsquo; reduction levels can indicate on good basis of health safe chicken meat production. Analysis of storage duration has shown that osmotdehydrated chicken meat is microbilogicaly and chemicaly stabile during sotrage at 22 &deg;C in period of at least 10 days.</p>

Listeria monocytogenes transporte le cellobiose principalement via le PTS (PEP:carbohydrate phosphotransferase system). La croissance sur cellobiose induit l'expression des opérons celBCA1, celBA2 ainsi que du gène lmrg_01989, qui codent respectivement le composant soluble EIIACel1, le transporteur EIICCel1, le composant soluble EIIBCel1, les protéines EIIBCel2 et EIIACel2, et une seconde EIICCel. La croissance sur glucose réprime fortement l'expression de ces gènes. La délétion de celC1 codant l'EIICCel1 ou des deux gènes, celA1 et celA2, ralentit considérablement la consommation cellobiose. L'expression des trois unités de transcription induite par le cellobiose dépend de CelR. CelR, qui code un régulateur transcriptionnel LevR- like, est situé en aval de l'opéron bicistronique celBA2. CelR est activé par phosphorylation par EI et HPr de l'His550. En revanche, la phosphorylation de l'His823, catalysée par P~EIIBCel1 et P~EIIBCel2, inhibe l'activité de CelR. Le remplacement de l'His823 par une Ala empêchant cette phosphorylation ou la délétion des deux gènes codants les EIIAsCel ou EIIBsCel entraîne l'expression constitutive des trois unités de transcription contrôlées par CelR. Comme le glucose, le cellobiose inhibe fortement l'activité de PrfA, l'activateur des gènes de virulence. Nous avons donc cherché à tester si l'un des composants PTSCel pouvait être impliqué dans la répression de gènes de virulence. Les mutants consommant faiblement le cellobiose, présentaient une levée de la répression des gènes de virulence par le cellobiose, alors que le glucose et les autres sucres-PTS les réprimaient toujours. De manière surprenante, la délétion du gène monocistronique lmrg_00557, qui code un autre composant EIIBCel du PTS, induisait la levée de la répression des gènes de virulence médiée par toutes les sources de carbone mais n'avait aucun effet sur la consommation de glucose ou de cellobiose. Ce gène lmrg_00557 a été appelé vgiB (virulence gene inhibitor B) et la protéine correspondante, qui semble jouer un rôle majeur dans la régulation de l'activité de PrfA, EIIBVir. Cette protéine est phosphorylée par le PEP et les composants PTS EI, HPr et EIIACel2 sur le résidu cystéine-8. La complémentation du mutant ΔvgiB avec l'allèle sauvage, mais également avec l'allèle Cys8Ala, restaurait le mécanisme général de répression des gènes de virulence par les sucres, suggérant ainsi que la forme non phosphorylée de EIIBVir inhibe l'activité de PrfA. / Listeria monocytogenes transports cellobiose mainly via a PEP:carbohydrate phosphotranseferase system (PTS). Growth on cellobiose induces the expression of the celBCA1 and celBA2 operons as well as lmrG01989, which encode the soluble EIIA Cel1 and EIIB Cel1 components, the transporter EIIC Cel1 , the EIIA Cel2 and EIIB Cel2 proteins, and a second EIIC Cel , respectively. Growth on lucose strongly repressed the expression of these genes. Deletion of the EIIC Cel1 –encoding celC1 or of both, celA1 and celA2, significantly slowed cellobiose consumption. The bicistronic operon celBA2 is located downstream from celR, which codes for a LevR-like transcription activator. Expression of the three cellobiose-induced transcription units depends on CelR. The gene encoding CelR is located upstream from the bicistronic operon celBA2. CelR itself is activated via phosphorylation by EI and HPr at His550. In contrast, phosphorylation at His823, which is catalyzed by both, P~EIIB Cel1 and P~EIIB Cel2 , inhibits CelR activity. Preventing this phosphorylation by replacing His823 with Ala or deleting the two EIIA Cel – or EIIB Cel -encoding genes caused constitutive expression of all three CelR-controlled transcription units. Similar to glucose, cellobiose strongly inhibits the activity of the virulence gene activator PrfA. We therefore tested whether one of the PTS Cel components might be involved in virulence gene repression. Mutants, that exhibit slow cellobiose consumption, were relieved from cellobiose-mediated virulence gene repression, whereas glucose and other PTS-sugars still repressed them. Strikingly, deletion of the presumed monocistronic lmrg_00557, which codes for another EIIB Cel -like PTS component, caused a general relief from carbon source-mediated virulence gene repression, but had no effect on cellobiose or glucose consumption. The gene lmrg_00557 was named vgiB (virulence gene inhibitor B) and the encoded protein, which seems to play a major role in PrfA regulation, was called EIIB Vir . It becomes phosphorylated by PEP and the PTS components enzyme I, HPr and EIIA Cel2 at cysteine-8. Complementation of the ΔvgiB mutant with wild-type vgiB, but also with the Cys8Ala allele restored general virulence gene repression, thus suggesting that it is the unphosphorylated form of EIIB Vir , which inhibits the activity of PrfA.

Master of Science / Food Science Institute -- Animal Science & Industry / Daniel Y.C. Fung / In February 2008, the FDA released a draft Compliance Policy Guide (CPG) on Listeria monocytogenes and proposed that ready-to-eat (RTE) foods that do not support the growth of L. monocytogenes may contain up to 100 CFU/g of this pathogen. Frozen foods such as ice cream fall in that category since they are consumed in the frozen state. However, other frozen foods, such as vegetables and seafood that are thawed and served at salad and food bars, may support the growth of Listeria and would not be allowed to contain 100 CFU/g according to the draft CPG. In the current study, growth curves were generated for L. monocytogenes inoculated onto four thawed frozen foods - corn, green peas, crabmeat, and shrimp - stored at 4, 8, 12, and 20ºC. Growth parameters, lag phase duration (LPD), and exponential growth rate (EGR) were determined using a two-phase linear growth model and the Square Root Model. The results demonstrated that L. monocytogenes has a very short LPD on these thawed frozen foods during refrigerated storage and that there would be several orders of magnitude of growth (i.e., more than 1.7 log increase at 4 ºC) of the organism before the product is found to be organoleptically unacceptable. Although it would not be possible to take advantage of any extended lag phase duration caused by freeze injury to the organism, frozen foods containing less than 100 CFU/g of L. monocytogenes that are thawed, or thawed and cooked, and then consumed immediately, should not represent a public health hazard.

Master of Science / Food Science / Kelly J.K. Getty / Listeria monocytogenes has the potential to contaminate ready-to-eat (RTE) meat and poultry products. Listeria monocytogenes contamination is a hazard that can potentially occur after post-lethality treatment in a processing environment during slicing or packaging of RTE meat products. United States Department of Agriculture’s Food Safety and Inspection Service (USDA/FSIS) requires facilities to have intervention strategies to demonstrate control of this pathogen in RTE meat and poultry products. FSIS categorizes different intervention strategies into Alternative 1, 2, or 3. If an establishment chooses Alternative 1, it must use a post-lethality treatment that reduces or eliminates microorganisms on the product and an antimicrobial agent or process that suppresses or limits the growth of L. monocytogenes. If an establishment chooses Alternative 2, it can either use a post-lethality treatment or an antimicrobial agent or process that suppresses or limits growth of L. monocytogenes. Under Alternative 3, the establishment must have a detailed sanitation program as its intervention strategy. As establishments increase the number of interventions or change from Alternative 3 to 2 to 1, the frequency of FSIS sampling of RTE meat and poultry products for safety and wholesomeness decreases. The effectiveness of post-package decontamination technologies such as high-pressure processing, ultraviolet C light, and pre/post-package surface pasteurization have been researched for controlling L. monocytogenes in RTE products. Formulating meat products with antimicrobial additives such as lactates, sodium lactate and sodium diacetate, potassium lactate and sodium diacetate, sodium levulinate, lauric arginate, glucono-delta-lactone, or organic acids is another common approach to control L. monocytogenes in RTE meat products. Also, a combination of sodium lactate and sodium diacetate in a formulation is an acceptable antimicrobial strategy to provide Alternative 2 status. Bacteriocins such as nisin can also be added to the formulation of RTE meat and poultry products for controlling L. monocytogenes. In addition nisin can be applied as packaging film coating. Another approach for controlling L. monocytogenes in products such as jerky, kippered steaks, snack sticks and turkey tenders is the use of packaging environments and holding times prior to shipping. In conclusion, there are various approaches for controlling L. monocytogenes in RTE meat and poultry products post-lethality and processors should consider these options rather than relying on sanitation alone.

Doctor of Philosophy / Food Science Institute / Elizabeth A. E. Boyle / A total of three studies were conducted to evaluate the effects of different packaging systems and storage times on reduction of Listeria monocytogenes on ready-to-eat meat snacks. Study 1 was conducted to determine the effects of four packaging systems [heat sealed (HS), heat sealed with oxygen scavenger (HSOS), nitrogen flushed with oxygen scavenger (NFOS), and vacuum (VAC)] and storage times (24, 48, and 72 h, and 14 and 30 d) on reduction of L. monocytogenes in turkey jerky in the presence or absence of sodium nitrite. Inclusion of sodium nitrite in turkey jerky did not affect (P>0.05) L. monocytogenes log reductions regardless of packaging type or storage time. After 14 d of storage in HSOS, NFOS, or VAC, and 48 or 72 h in HS, a reduction of >1.0 log CFU/cm² of L. monocytogenes was achieved. Processors could use HS in conjunction with 48 h of ambient storage and be in compliance with the United States Department of Agriculture Food Safety and Inspection Service Listeria Rule of post-lethality treatment in achieving at least 1 log reduction of L. monocytogenes. Study 2 was conducted to investigate attachment of L. monocytogenes to uncured and cured turkey jerky packaged in HS, HSOS, NFOS, or VAC using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The SEM examination showed that L. monocytogenes is capable of adhering to uncured or cured turkey jerky surfaces. Elemental maps from EDS analysis revealed that no element was unique or elevated at sites of L. monocytogenes attachment. Elemental composition showed the presence of elemental sulfur and could be an indication of the presence of sulfur-containing amino acids in turkey jerky. Finally, Study 3 evaluated the affects of two packaging types (HSOS and NFOS) and four ambient storage times (24, 48, and 72 h, and 14 d) on reduction of L. monocytogenes on five commercial RTE meats and poultry snacks (beef tenders, beef jerky, beef sausage sticks, pork jerky, and turkey sausage sticks). A mean reduction of >1.0 log CFU/cm² of L. monocytogenes was achieved on all products, regardless of packaging or storage time. Correlation analysis provided some indication that reduction of L. monocytogenes increased with fat content. However, the strength of linear correlation was not sufficient to account for the differences in log reduction in L. monocytogenes. In study 1, a holding time of 24, 48, or 72 h for HSOS or NFOS packaging of was not effective for reducing L. monocytogenes by at least 1 log on turkey jerky. In contrast, packaging beef tenders, beef jerky, beef sausage sticks, pork jerky, and turkey sausage sticks in HSOS or NFOS for at least 24 h ambient storage was sufficient to achieve at least 1 log reduction in L. monocytogenes population. Specific components such as sulfur-containing amino acids in turkey jerky might be contributing to <1 log reduction of L. monocytogenes population on turkey jerky after 24, 48, or 72 h of ambient storage. Overall, nitrite was not an effective ingredient to control L. monocytogenes in turkey jerky. However, packaging such as HS, HSOS, NFOS or VAC and at least 24 h holding time were effective hurdles for controlling L. monocytogenes at post-lethality.