O presunto cozido é um dos embutidos cárneos mais consumidos no Brasil, sendo também um dos mais sensíveis à deterioração por bactérias ácido láticas (BAL) e a contaminação por Listeria monocytogenes. O objetivo deste trabalho foi avaliar a atividade antimicrobiana da nisina (Nisaplin®) aplicada em presunto cozido, a fim de controlar L. monocytogenes e BAL. Para tanto, presuntos cozidos foram preparados e fatiados individualmente pelas empresas DuPont e Marsul. No ensaio 1, o presunto foi injetado com salmoura contendo 12,5 mg de nisina por kg (dosagem aprovada no Brasil para queijos fundidos). O potencial bioconservante da nisina foi avaliado contaminando artificialmente as fatias do presunto com um pool composto por 5 cepas de L. monocytogenes. Os resultados do ensaio 1 demonstraram que o tratamento com nisina não teve efeito significativo sobre a população de L. monocytogenes. Já as BAL foram inibidas por 2 dias. No ensaio 2, a adição da nisina na mesma dosagem foi feita no tambleamento, inibindo a população de L. monocytogenes por 6 dias e BAL por 10 dias Em vista dos resultados obtidos nos 2 primeiros ensaios foi determinada a concentração mínima bactericida (CMB) do pool e dos isolados de L. monocytogenes, a fim de verificar a sensibilidade das cepas individualmente e em conjunto e o efeito da nisina sem a matriz cárnea. Além disso, também foi avaliado o efeito sinérgico ou antagônico da salmoura sobre a ação bactericida da nisina. Pôde-se constatar que as diferentes cepas de L. monocytogenes demonstraram perfis de sensibilidade diferentes e que o pool de cepas foi menos sensível ao efeito da nisina. Os resultados obtidos conduziram a avaliação de dosagem maior de nisina no presunto cozido. No ensaio 3, 32 mg/kg de nisina foram adicionadas a salmoura do presunto cozido e testados contra o pool de L. monocytogenes e a cepa ATCC 7644, separadamente. Os resultados demonstraram que a nova dosagem de nisina inibiu significativamente a multiplicação do pool de L. monocytogenes e da cepa ATCC 7644, durante 10 dias, sugerindo ser uma barreira efetiva no controle de L. monocytogenes em presunto cozido. / Precooked ham is one of the most consumed meats in Brazil. It is also one of the most sensitive to spoilage by lactic acid bacteria (LAB) and contamination by Listeria monocytogenes. The objective of this study is to evaluate the antimicrobial activity of Nisin (Nisaplin®) when applied to precooked ham, in order to control the growth of L. monocytogenes and LAB. For the purposes of this study, the precooked ham prepared and sliced individually by the DuPont and Marsul company was used. For trial 1, brine containing 12.5 mg of nisin was injected per kg of ham (dosage approved in Brazil for processed cheese). The biopreservative potential of nisin was evaluated by artificially contaminating slices of control and test ham with a pool of 5 different strains of L. monocytogenes. The results of this first trial showed that treatment with this amount of nisin had no significant effect on the growth of the L. monocytogenes population. However, the LAB population was indeed inhibited by 2 days. In trial number 2, the addition of nisin to the same amount of precooked ham by tumbling inhibited the growth of the L. monocytogenes population by 6 days and of the LAB population by 10 days. In light of the results obtained in these first 2 trials, the minimum bactericidal concentration (MBC) pool and L. monocytogenes isolates were determined in order to verify the sensitivity of the strains individually and together as well as the effects of nisin without the meat matrix Additionally, the synergistic or antagonistic effects of brine on the antimicrobial properties of nisin were also evaluated. Results suggest that different strains of L. monocytogenes have different sensitivity profiles and that the pool of strains was less sensitive to the effects of the nisin. These results prompted an increase in the dosage of nisin added to the precooked ham in trial number 3. In this trial, 32mg/kg of nisin was added to the brine of the precooked ham and tested against the L. monocytogenes pool and ATCC 7644 strain, separately. Results demonstrated that this increase in amount of nisin significantly reduced the growth of the L. monocytogenes pool and of the ATCC strain for 10 days. This suggests that nisin can be an effective barrier against the growth of L. monocytogenes in precooked ham.

O objetivo do presente estudo foi monitorar a incidência de Staphylococcus aureus, Listeria sp., Listeria monocytogenes, Escherichia coli, coliformes totais, bactérias aeróbias mesófilas e psicrotróficas ao longo da produção de leite pasteurizado, desde a ordenha até a obtenção do produto final para estabelecer etapas e locais críticos da produção, bem como avaliar se a presença de Listeria sp. constitui um bioindicador de L. monocytogenes, e E. coli um bioindicador de outros micro-organismos patogênicos. As coletas foram feitas em 5 laticínios (A, B, C, D e E) do Estado de São Paulo, em duplicata, com intervalo de coleta variando de 3 semanas a 7 meses, de acordo com disponibilidade dos laticínios. Coletou-se um total de 236 amostras foram coletadas, sendo 36 de leite (cru e pasteurizado), 162 eram provenientes de superfícies que não tinham contato com o leite e 38 superfícies que entravam em contato com leite. Das 36 amostras de leite analisadas, 13,9% estavam contaminadas com Listeria sp. e nenhuma com L. monocytogenes; 61,1% continham E. coli e 5,6% apresentavam S. aureus. Somente o leite do laticínio C apresentou em uma das coletas micro-organismo patogênico (E. coli) no leite pasteurizado, indicando falhas no processamento ou no manejo no momento da ordenha. Das 38 amostras de superfícies com contato com o leite (38), 2,6% foram positivas para Listeria sp., 50,0% para E. coli e 5,3% para S. aureus. Das amostras de superfícies sem contato com o leite (162), 13,3% estavam contaminas com Listeria sp., 6,2% com L. monocytogenes e 25,9% com E. coli. De acordo com o limite estabelecido de aeróbios mesófilos no leite cru pela IN 62, constatou-se que 50,0% do leite cru dos laticínios A, D e E, 100% do leite cru do laticínio B e 33,3% do leite cru do laticínio C estão fora dos padrões estabelecidos pela legislação. Foi comprovado que a Listeria sp. não pode ser considerada como bioindicador de L. monocytogenes pelo teste Qui-Quadrado (p<0,05). Ao comparar as médias das amostras positivas para os microorganismos E. coli, S. aureus, Listeria sp. e L. monocytogenes dos laticínios processadores de leite tipo A com os de leite pasteurizado, somente o S. aureus no leite apresentou diferença significativa pelo teste \"T\" (p<0,05). Além dos pontos críticos de controle (PCC) checados através da Árvore Decisória (pasteurização, superfícies internas de embalagens), outros pontos merecem destaque pela elevada quantidade de patógenos (tanques de armazenamento de leite cru e pisos e paredes de câmaras frias). Os resultados obtidos ressaltam a importância da adoção de ferramentas de gestão da qualidade, como Boas Práticas de Fabricação e APPCC, para que a segurança alimentar seja garantida ao longo da cadeia de produção do leite pasteurizado nos laticínios estudados. / The aim of this study was to monitor the incidence of Staphylococcus aureus, Listeria sp., Listeria monocytogenes, Escherichia coli, total coliforms, mesophilic aerobic and psychrotrophic bacteria along the pasteurized milk production, from milking to the final product, to establish steps and critical points of production, as well as to evaluate the presence of Listeria sp. as a bioindicator of L. monocytogenes and E. coli a bioindicator of other pathogenic microorganisms. Duplicate samples were collected in 5 dairy plants (A, B, C, D, E) from the state of São Paulo, within intervals ranging from 3 weeks to 7 months, according to the dairy plants availability. A total of 236 samples were collected, being 36 of milk (raw and pasteurized), 162 from surfaces with no contact with the milk, and 38 from surfaces with contact with milk. Out of 36 milk samples analyzed, 13.9% were contaminated with Listeria sp. and none had L. monocytogenes; 61.1% were contaminated with E. coli and 5.6% with S. aureus. Only dairy plant C showed pathogenic microorganism (E. coli) in the pasteurized milk in one of the collections, indicating failures in the pasteurization or excessive bacterial load in the raw milk. Out of the 38 samples of surfaces that had contact with milk, 2.6% were positive for Listeria sp., 50.0% for E. coli and 5.3% for S. aureus. As for the samples from surfaces with no contact with milk (162), 13.3% were contaminated with Listeria sp., 6.2% with L. monocytogenes and 25.9% with E. coli. According to the Brazilian regulations for aerobic mesophiles in raw milk by Normative Instruction 62, 50.0% of samples from dairy plants A, D and E, 100% of samples from dairy plant B and 33.3% of samples from dairy plant C were above the tolerance limit adopted. The analysis of Listeria sp. could not be considered as a bioindicator of L. monocytogenes by chi-square test (p<0.05). When comparing the mean frequencies of positive samples for E. coli, S. aureus, Listeria sp. and L. monocytogenes in the processing dairy plants of type A milk (plants A and B) and the pasteurized one (plants C, D and E), only S. aureus in milk showed significant difference by \"T\" test (p<0.05). In addition to the critical control points (CCP) checked by a decision tree (pasteurization, internal surfaces of packaging), other points should be highlighted by the high number of pathogens found (bulk raw milk tanks, floors and walls of cold storage rooms). Results of this trial indicate the importance of adoption of quality management tools such as Good Manufacture Practices and HACCP, to ensure food safety along the pasteurized milk production chain in the dairy plants evaluated.

Listeria monocytogenes é um micro-organismo Gram-positivo que está comumente associado a doenças de origem alimentar. Possui a capacidade de sobreviver a condições adversas e de formar biofilme em diferentes superfícies abióticas, tornando-se um problema constante para a indústria de alimentos, pois pode comprometer a sanitização e aumentar o risco de contaminação pós-processamento. A formação de biofilme pode ser regulada por um mecanismo denominado quorum sensing, no qual ocorre intensa comunicação célula-célula, mediada por moléculas químicas, chamadas de autoindutoras. Pouco se sabe sobre a ocorrência de interação entre bactérias Gram- positivas e negativas na formação de biofilmes, sendo mais frequentes estudos entre bactérias do mesmo grupo. A fim de avaliar a ocorrência de interação entre Escherichia coli e L. monocytogenes (Lm), desenvolveu-se esta pesquisa com os seguintes objetivos: i) verificar a capacidade de Lm sorotipo 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) formar biofilme em presença de Escherichia coli, avaliando-se a importância dos reguladores de virulência, prfA e sigB, no processo; e ii) verificar a produção e interferência de moléculas autoindutoras de E. coli E2348/69 na formação de biofilme por Lm. Os ensaios de formação de biofilme foram realizados utilizando-se lâminas de aço-inoxidável AISI 304 #4 imersas em caldo infusão de cérebro e coração (BHI) e em meio pré-condicionado (MPC) por E. coli, com incubação a 25 ºC. Foram testadas duas concentrações iniciais de Lm (102 e 106 UFC.mL-1) e amostragens em diferentes tempos de incubação. Utilizou-se um método de quantificação indireto com coloração do biofilme por cristal violeta e posterior leitura da absorbância. Observou-se que Lm 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) são capaz de formar biofilme na presença de Escherichia coli e que uma maior quantidade de biofilme foi formada por Lm selvagem quando comparada à sua mutante, em meio não pré-condicionado (controle), indicando que prfA e sigB estão envolvidos no processo de formação de biofilme. Quando em MPC, o biofilme formado pela cepa selvagem foi menor que no meio controle (BHI), indicando que E. coli E2348/69, utilizada no pré-condicionamento do meio, produz moléculas capazes de interferir no processo de formação e na quantidade de biofilme formado por Lm; e para o biofilme formado pela cepa mutante, houve uma maior quantificação em MPC em comparação ao meio controle, o que sugere que os genes deletados possam estar envolvidos no reconhecimento das moléculas autoindutoras. Assim, os dados obtidos permitem concluir que há interação e interferência por parte de E. coli na formação de biofilme por Lm mediante produção de moléculas autoindutoras. / Listeria monocytogenes (Lm) is a Gram-positive microorganism commonly associated with foodborne diseases. Due to its ability to survive under adverse environmental conditions and to form biofilm in different abiotic surfaces, this bacterium is a concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Biofilm formation can be regulated by a quorum sensing mechanism, in which there is intense cell-cell communication mediated by chemical molecules, called autoinducers. Little is known about the occurrence of interaction between Gram-positive and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate the occurrence of interaction between Escherichia coli and Lm, this study was developed including the following objectives: i) to evaluate the ability of Lm 1/2a and its isogenic mutant strain (ΔprfAΔsigB) to form biofilm on the presence of Escherichia coli, assessing the importance of virulence regulators, prfA and sigB, in this process; and ii) to verify the production and interference autoinducers of E. coli E2348/69 on biofilm formation by Lm. Biofilm formation assays were conducted using stainless steel AISI 304 #4 immersed into broth brain heart infusion (BHI) and into preconditioned medium (MPC) by E. coli, following incubation at 25 °C. Lm at two initial concentrations (102 and 106 CFU.mL-1) and under different incubation time was tested. An indirect method for quantification of cells was applied, using crystal violet to color the biofilm, followed by optical density measurement. It was observed that Lm 1/2a and its isogenic mutant (ΔprfA ΔsigB) are able to form biofilm in the presence of Escherichia coli and a larger amount of biofilm was formed by wild strain Lm compared to its mutant, in a non-preconditioned medium (control), indicating that prfA and sigB are involved in biofilm formation. For MPC, the biofilm formation by the wild strain was lower than in the control (BHI), indicating that E. coli E2348/69, used in the preconditioned medium, produces molecules that can affect the formation process and the amount of biofilm formed by Lm; and in the biofilm formed by the mutant strain, there was a higher quantification of MPC compared to the control, suggesting that the deleted genes may be involved in recognition the of autoinducers. These results suggest that there is an interaction and interference of E. coli on biofilm formation by Lm due the production of autoinducers.

A preferência por produtos prontos para consumo pode implicar em aumento do risco de doenças transmitidas por alimentos (DTAs) e uma grande preocupação, nesse caso, é a presença da Listeria monocytogenes. A infecção por essa bactéria apresenta baixa taxa de morbidade, porém alta de mortalidade, representando maior risco para gestantes, idosos, crianças e indivíduos imunodeprimidos. Os produtos considerados de maior risco são aqueles prontos para o consumo, mantidos sob refrigeração e com longa vida útil. Face ao exposto, foi pesquisada a ocorrência de L. monocytogenes em dois grupos de produtos cárneos fatiados: presunto cozido e salame. Cento e trinta amostras de cada tipo de produto, adquiridas no comércio varejista do Município de São Paulo, foram submetidas a análises laboratoriais. Tais análises foram conduzidas em dois momentos: no terço inicial e no final de vida útil dos produtos. Nos casos de positividade, foram realizadas a quantificação e a sorotipagem da bactéria em cada um dos produtos, a fim de avaliar se os resultados obtidos poderiam oferecer risco à saúde. O salame apresentou prevalência significativamente maior para a L. monocytogenes, 6,2 por cento (8/130), enquanto no presunto a prevalência foi de 0,8 por cento (1/130). As contagens nas amostras de salame apresentaram valores entre <10 a 1900 UFC/g. Os sorotipos identificados, considerando os dois tipos de produtos, apresentaram as seguintes freqüências: 4b= 37,5 por cento (3/8), 1/2b= 25 por cento (2/8), 3b= 25 por cento (2/8) e 1/2c= 12,5 por cento (1/8). Os resultados encontrados permitem inferir que, para os produtos analisados, o risco de listeriose decorrente do consumo de salame é maior do que o associado ao consumo de presunto cozido / The preference for ready-to-eat products can raise the risk of diseases transmitted by food and in this case there is a main concern about the presence of Listeria monocytogenes. The infection caused by these bacteria presents low morbidity but high mortality rate, representing higher risk to pregnants, elderly, children and immunodepressed people. Products considered to have higher risk are the ready-to-eat kept under refrigeration and with longer shelf life. Considering this, it has been searched the occurrence of L. monocytogenes in two groups of sliced meat: cooked jam and salami. There were submitted to laboratorial analyses, to identification of L. monocytogenes, 130 samples of each product, acquired from São Paulo retailing market. Analyses were conducted in two times, in the starting third part life of product and in the end of shelf live. For the positive cases it was realized quantification and serotype from this bacterium, in order to evaluate if found results can offer risk to health. Salami has presented occurrence significantly higher for L. monocytogenes, 6.2 per cent (8/130), while cooked jam has presented 0.8 per cent (1/130). Counts of salami have shown results from <10 to 1900 CFU/g. Identified serotypes, considering both types of products, presented the following frequencies: 4b= 37,5 per cent (3/8), 1/2b= 25 per cent (2/8), 3b= 25 per cent (2/8) e 1/2c= 12,5 per cent (1/8). Presented results allow us to infer, to the tested products, that the risk of listeriosis from consuming salami is higher than the risk associate to consuming cooked jam

Listeria monocytogenes é um importante patógeno de origem alimentar que causa listeriose, infecção severa que acomete, principalmente, gestantes, idosos, crianças e imunocomprometidos, e que apresenta elevada taxa de mortalidade. A bactéria está amplamente distribuída no ambiente e é comumente encontrada em produtos cárneos. O presente estudo teve como objetivos caracterizar 439 isolados de L. monocytogenes obtidos de salsicha bovina e produtos cárneos crus (carne moída, linguiça suína e coxa de frango) refrigerados, adquiridos no comércio do município de São Paulo, e previamente submetidos à sorotipagem molecular. Os isolados foram caracterizados quanto ao perfil de susceptibilidade antimicrobiana; presença dos genes de virulência actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB e mpl; perfil genético por eletroforese em campo pulsado (PFGE) e sequenciamento parcial dos genes actA e lmo0737. Baixa frequência de resistência antimicrobiana (0,5%) foi observada entre os 416 isolados avaliados. Um isolado pertencente ao sorogrupo 1 apresentou resistência à penicilina e à clindamicina e outro identificado como 4a ou 4c apresentou resistência à tetraciclina. Todos os isolados foram positivos para os genes de virulência testados. O sequenciamento parcial do gene actA mostrou a ocorrência de 14 sequências de nucleotídeos distintas nos 97 isolados avaliados. Além disso, verificou-se a ocorrência de uma deleção de 35 aminoácidos no gene actA em 36 isolados, além de substituições de nucleotídeos que resultaram em mutações nas sequências de aminoácidos da grande maioria dos isolados. A análise filogenética do gene actA possibilitou o agrupamento dos isolados em duas linhagens distintas (I e II). Os resultados do PFGE indicaram grande variabilidade nos perfis genéticos dos isolados analisados, principalmente naqueles pertencentes aos grupos 2 (1/2c e 3c), 3 (1/2b e 3b) e 4 (4b, 4d e 4e). Os resultados deste estudo mostram que os isolados de L. monocytogenes provenientes de salsicha bovina e produtos cárneos crus comercializados no município de São Paulo, apresentam grande diversidade genética, importante potencial de virulência e baixa frequência de resistência antimicrobiana. A diversidade observada deve-se, provavelmente, à característica ubíqua deste micro-organismo, tornando-o mais susceptível a grande pressão seletiva do ambiente. / Listeria monocytogenes is an important foodborne pathogen that causes listeriosis, a severe infection that affects primarily pregnant women, elderly, children and imunocompromised individuals, and has a high mortality rate. The bacteria is widely distributed in the environment and commonly found in meat products. The present study aimed to characterize 439 isolates of L. monocytogenes obtained from pork sausage and raw chilled meat products (ground beef, beef sausage, and chicken thigh) purchased in supermarkets in the city of São Paulo, and previously submitted to molecular serotyping. The isolates were characterized for antimicrobial susceptibility profile; presence of virulence genes actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB and mpl; genetic profile by pulsed field gel electrophoresis (PFGE) and partial sequencing of genes actA and lmo0737. A low frequency of antimicrobial resistance (0.5%) was observed among the 416 evaluated isolates. One isolate belonging to serogroup 1 presented resistance to clindamycin and penicillin and another one identified as 4a or 4c was resistant to tetracycline. All isolates were positive for the tested virulence genes. The partial sequencing of the gene actA indicated the occurrence of 14 distinct nucleotide sequences in the 97 isolates tested. Furthermore, a deletion of 35 amino acids in the actA gene was detected in 36 isolates, and nucleotide substitutions that resulted in amino acid changes in the sequences of most isolates. Phylogenetic analysis of the actA gene clustered the isolates in two distinct lineages (I and II). Results of PFGE indicated a great genetic variability among isolates, especially among those belonging to groups 2 (1/2c and 3c), 3 (1/2b and 3b) and 4 (4b, 4d and 4e). The results of this study show that isolates of L. monocytogenes from pork sausage and raw meat products marketed in the city of São Paulo present a great genetic diversity, significant virulence potential and low frequency of antimicrobial resistance. The detected diversity is probably due the ubiquitous nature of these microorganisms, making them more susceptible to selective pressure of the environment.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

A produção de leite e de seus derivados lácteos geram grande impacto no setor agroindustrial. Classificado como um dos alimentos mais completos, o leite é rico em nutrientes essenciais ao crescimento e à manutenção de uma vida saudável. Em contrapartida, os constituintes do leite propiciam excelente meio de cultura para o desenvolvimento de microrganismos, desde o momento da ordenha até a chegada ao consumidor final. Neste contexto, estudos acerca de novas alternativas que auxiliem no controle da qualidade do leite são essenciais. A fotoinativação com luz azul tem surgido como uma alternativa antimicrobiana em potencial na indústria alimentícia, pois exerce atividade antimicrobiana intrínseca, sem o envolvimento de substâncias fotossensibilizadoras exógenas. O objetivo deste estudo foi determinar a efetividade da fotoinativação por luz azul (LED azul, &lambda;&#61; 410 ± 15 nm, 100 mW) sobre Staphylococcus aureus, Escherichia coli, Listeria monocytogenes e Mycobacterium fortuitum suspensos em leite integral UHT e solução PBS. Os resultados demonstraram a fotinativação de todas as cepas empregadas no estudo pela luz azul, em tempos distintos, independentemente do meio utilizado, isto é, M. fortuitum, S. aureus, E. coli e L. monocytogenes apresentaram cinética de fotoinativação diferentes entre si, quando suspensos no leite ou no PBS (p<0,0001). O fator de resistência R foi menor que 1 (R < 1) em meio leite, sendo 0,82, 0,78, 0,88 e 0,81 para S.aureus, E. coli, L. monocytogenes e M. fortuitum, nesta ordem, apresentando-se, portanto, mais sensíveis à luz azul nos tempos iniciais e ocorrendo fotoinativação em maior proporção neste período. Quando suspensos em PBS, à semelhança do meio leite, L. monocytogenes e E. coli, apresentaram valor de R < 1 (0,87 e 0,81, respectivamente), ao passo que S. aureus e M. fortuitum apresentaram R > 1, isto é, 1,06 e 1,46, respectivamente, demonstrando maior resistência à fotoinativação nos períodos iniciais e tornando-se mais sensíveis conforme o tempo de irradiação progredia. Concluiu-se que a luz azul foi capaz de fotoinativar, in vitro, cepas de S. aureus, E. coli, L. monocytogenes e M.fortuitum, suspensas em PBS e em leite integral UHT. A fotoinativação com luz azul apresenta caráter inovador, sendo uma alternativa potencialmente promissora no controle da contaminação por microrganismos na indústria láctea / The production of milk and its dairy products have great impact on the agro-industrial sector. Considered as one of the most complete foods, milk is full of essential nutrients required for growth and maintenance of a healthy life. On the other hand, the composition of milk makes it an excellent growth medium for many microorganisms, from the moment of milking to its arrival to the final consumer. In this context, studies toward the search for new alternatives are essential for milk quality improvement. The photoinactivation by blue light has emerged as an alternative antimicrobial approach in the food industry due to its intrinsic antimicrobial properties without the involvement of exogenous photosensitizers. The aim of this study was to assess the effectiveness of blue light photoinactivation (blue LED, &lambda; &#61; 410 ± 15 nm, 100 mW) on Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Mycobacterium fortuitum strains suspended in UHT whole milk and PBS medium. All used strains was sucesssfully photoinactivated by the blue light, at different moments, regardless of the medium used, i.e., M. fortuitum, S. aureus, E. coli and L. monocytogenes presented different photoinactivation kinetics when suspended in the UHT whole milk or PBS (p < 0.0001). The resistance R factor was less than 1 (R < 1) in milk medium, with 0.82, 0.78, 0.88 and 0.81 for S. aureus, E. coli, L. monocytogenes and M. fortuitum, respectively, meaning that the strains were most sensitive to blue light in the initial times, when the photoinactivation was higher. When suspended in PBS, as occurred in the the milk medium, L. monocytogenes and E. coli presented R < 1 (0.87 and 0.81, respectively), whereas S. aureus and M. fortuitum demonstrated R > 1, i.e., 1.06 and 1.46, respectively, indicating higher photoinactivation resistance in the initial stages and higher sensibility as the irradiation time progressed. We concluded that blue light promoted in vitro photoinactivation of the strains of S. aureus, E. coli, L. monocytogenes and M.fortuitum, suspended in PBS and UHT whole milk. The photoinactivation by blue light presents an innovative approach and a promising and exciting alternative for microbial contamination control in dairy industry

Listeria monocytogenes é um importante patógeno de origem alimentar que causa listeriose, infecção severa que acomete, principalmente, gestantes, idosos, crianças e imunocomprometidos, e que apresenta elevada taxa de mortalidade. A bactéria está amplamente distribuída no ambiente e é comumente encontrada em produtos cárneos. O presente estudo teve como objetivos caracterizar 439 isolados de L. monocytogenes obtidos de salsicha bovina e produtos cárneos crus (carne moída, linguiça suína e coxa de frango) refrigerados, adquiridos no comércio do município de São Paulo, e previamente submetidos à sorotipagem molecular. Os isolados foram caracterizados quanto ao perfil de susceptibilidade antimicrobiana; presença dos genes de virulência actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB e mpl; perfil genético por eletroforese em campo pulsado (PFGE) e sequenciamento parcial dos genes actA e lmo0737. Baixa frequência de resistência antimicrobiana (0,5%) foi observada entre os 416 isolados avaliados. Um isolado pertencente ao sorogrupo 1 apresentou resistência à penicilina e à clindamicina e outro identificado como 4a ou 4c apresentou resistência à tetraciclina. Todos os isolados foram positivos para os genes de virulência testados. O sequenciamento parcial do gene actA mostrou a ocorrência de 14 sequências de nucleotídeos distintas nos 97 isolados avaliados. Além disso, verificou-se a ocorrência de uma deleção de 35 aminoácidos no gene actA em 36 isolados, além de substituições de nucleotídeos que resultaram em mutações nas sequências de aminoácidos da grande maioria dos isolados. A análise filogenética do gene actA possibilitou o agrupamento dos isolados em duas linhagens distintas (I e II). Os resultados do PFGE indicaram grande variabilidade nos perfis genéticos dos isolados analisados, principalmente naqueles pertencentes aos grupos 2 (1/2c e 3c), 3 (1/2b e 3b) e 4 (4b, 4d e 4e). Os resultados deste estudo mostram que os isolados de L. monocytogenes provenientes de salsicha bovina e produtos cárneos crus comercializados no município de São Paulo, apresentam grande diversidade genética, importante potencial de virulência e baixa frequência de resistência antimicrobiana. A diversidade observada deve-se, provavelmente, à característica ubíqua deste micro-organismo, tornando-o mais susceptível a grande pressão seletiva do ambiente. / Listeria monocytogenes is an important foodborne pathogen that causes listeriosis, a severe infection that affects primarily pregnant women, elderly, children and imunocompromised individuals, and has a high mortality rate. The bacteria is widely distributed in the environment and commonly found in meat products. The present study aimed to characterize 439 isolates of L. monocytogenes obtained from pork sausage and raw chilled meat products (ground beef, beef sausage, and chicken thigh) purchased in supermarkets in the city of São Paulo, and previously submitted to molecular serotyping. The isolates were characterized for antimicrobial susceptibility profile; presence of virulence genes actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB and mpl; genetic profile by pulsed field gel electrophoresis (PFGE) and partial sequencing of genes actA and lmo0737. A low frequency of antimicrobial resistance (0.5%) was observed among the 416 evaluated isolates. One isolate belonging to serogroup 1 presented resistance to clindamycin and penicillin and another one identified as 4a or 4c was resistant to tetracycline. All isolates were positive for the tested virulence genes. The partial sequencing of the gene actA indicated the occurrence of 14 distinct nucleotide sequences in the 97 isolates tested. Furthermore, a deletion of 35 amino acids in the actA gene was detected in 36 isolates, and nucleotide substitutions that resulted in amino acid changes in the sequences of most isolates. Phylogenetic analysis of the actA gene clustered the isolates in two distinct lineages (I and II). Results of PFGE indicated a great genetic variability among isolates, especially among those belonging to groups 2 (1/2c and 3c), 3 (1/2b and 3b) and 4 (4b, 4d and 4e). The results of this study show that isolates of L. monocytogenes from pork sausage and raw meat products marketed in the city of São Paulo present a great genetic diversity, significant virulence potential and low frequency of antimicrobial resistance. The detected diversity is probably due the ubiquitous nature of these microorganisms, making them more susceptible to selective pressure of the environment.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.