Efeito e modo de ação das bacteriocinas produzidas por Lactococcus lactis subsp. lactis ITAL 383, ATCC 11454 e CNRZ 150 contra Listeria innocua LIN 11

O efeito e o modo de ação das bacteriocinas produzidas por L. lactis subsp. lactis ITAL 383 e CNRZ 150 são similares à nisina de L. lactis subsp. lactis ATCC 11454. Estas bacteriocinas apresentaram um modo de ação bactericida, causando a lise de células de L. innocua LIN 11, associada ao decréscimo da absorbância e da viabilidade celular. O efeito letal foi maior para células em fase exponencial comparativamente à fase estacionária de crescimento. A adsorção dessas bacteriocinas às células de L. innocua LIN 11 foi muito rápida e influenciada pelo pH do meio de suspensão; adsorção máxima foi verificada a pH 6,0 e logo após o contato inicial. Perda completa de adsorção ocorreu em pH 2,0.

Modeling the variability of single-cell lag times for Listeria innocua populations after sublethal and lethal heat treatments

Optical density measurements were used to estimate the effect of heat treatments on the single-cell lag times of Listeria innocua fitted to a shifted gamma distribution. The single-cell lag time was subdivided into repair time ( the shift of the distribution assumed to be uniform for all cells) and adjustment time (varying randomly from cell to cell). After heat treatments in which all of the cells recovered (sublethal), the repair time and the mean and the variance of the single-cell adjustment time increased with the severity of the treatment. When the heat treatments resulted in a loss of viability (lethal), the repair time of the survivors increased with the decimal reduction of the cell numbers independently of the temperature, while the mean and variance of the single-cell adjustment times remained the same irrespective of the heat treatment. Based on these observations and modeling of the effect of time and temperature of the heat treatment, we propose that the severity of a heat treatment can be characterized by the repair time of the cells whether the heat treatment is lethal or not, an extension of the F value concept for sublethal heat treatments. In addition, the repair time could be interpreted as the extent or degree of injury with a multiple-hit lethality model. Another implication of these results is that the distribution of the time for cells to reach unacceptable numbers in food is not affected by the time-temperature combination resulting in a given decimal reduction.

Characterization of atypical Listeria innocua isolated from swine slaughterhouses and meat markets

Atypical Listeria innocua strains presenting phenotypic characteristics similar to those of Listeria monocyto genes were recently isolated from food and the environment. These isolates also tested positive for virulence genes specific to L. monocytogenes. Here we report the isolation of atypical hemolytic L. innocua strains from the environment of pork processing plants in Brazil. The strains were positive for L. monocytogenes virulence genes hly, inlA and inlB by PCR and presented genotypic similarities with human isolates of L. monocytogenes via the AFLP technique using HindIII single enzyme protocol. Phenotypic and genotypic similarities suggest that these atypical L. innocua may be pathogenic strains. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Microbiological quality of ready-to-eat minimally processed vegetables consumed in Brazil

Minimally processed leafy vegetables are ready-to-eat (RTE) products very attractive to consumers looking for healthy and convenient meals. However, the microbiological safety of these foods is of special concern due to the absence of lethal treatments during processing. In the present study, indicator microorganisms, Listeria spp. and Salmonella spp. were determined for 162 samples of minimally processed leafy vegetables commercialized in Brazil. Psychrotrophic aerobic bacterial populations >5 log CFU/g were found in 96.7% of the samples, while total and thermotolerant coliforms were detected respectively in 132 (81.5%) and 107 (66%) of vegetables analyzed. Escherichia coil was present in 86 (53.1%) samples analyzed and Listeria spp. and Salmonella spp. were detected respectively in 6 (3.7%) and 2 (1.2%) samples. These results indicate the need of implementing quality programs in the production chain of RTE vegetables to improve shelf life and microbiological safety. (C) 2011 Elsevier Ltd. All rights reserved.

An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage F serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages. (C) 2003 Elsevier B.V. All fights reserved.

Quantification of Listeria spp. contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon)

Aims: To quantify Listeria levels on the shell and flesh of artificially contaminated cooked prawns after peeling, and determine the efficacy of Listeria innocua as a model for L. monocytogenes in this system. Methods and Results: A L. monocytogenes and L. innocua strain were inoculated separately onto cooked black tiger prawns using two protocols ( immersion or swabbing with incubation). Prawns were peeled by two methods ( gloved hand or scalpel and forceps) and numbers of Listeria on shells, flesh and whole prawn controls were determined. Prawns were exposed to crystal violet dye to assess the penetration of liquids. Regardless of preparation method or bacterial strain there were ca 1log(10) CFU more Listeria per shell than per peeled prawn. Dye was able to penetrate to the flesh in all cases. Conclusions: Shell-on prawns may be only slightly safer than shell-off prawns. Listeria innocua is an acceptable model for L. monocytogenes in this system. Significance and Impact of the Study: Reduced risk from L. monocytogenes on prawns can only be assured by adequate hygiene or heating.

Formulação de um biofilme para controlo da listéria em queijos

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 12 Fevereiro de 2016, Universidade dos Açores.

Evaluation of phenotypic markers associated with pathogenicity in the genus Listeria

A total of 130 Listeria strains were tested in order to evaluate lecithinase production and capacity for Congo red adsorption as markers of pathogenicity. The strains were identified according to acid production from sugars and by the CAMP test and the data were correlated with the ability to produce keratoconjunctivitis in guinea pigs. L. monocytogenes cultures presented 51.8% and 88.8% positivity rates for Congo red adsorption and lecithinase production, respectively, whereas 80.8% and 100% for L. innocua cultures were negative for the two test, respectively

Surto de listeria monocytogenes na Região de Lisboa e Vale do Tejo : 2009-2011

RESUMO - O género Listeria contém oito espécies (L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, e a L. rocoutiae), das quais duas são patogénicas. L. monocytogenes é patogénica para humanos e animais; L. ivanovii primeiramente infecta animais e raramente causa doenças em humanos. A Listeria monocytogenes é uma bactéria patogénica Gram-positiva facultativa intracelular, ubíqua na natureza. Nos últimos anos o número de casos de listeriose tem vindo a aumentar. Pode causar uma doença rara e grave chamada listeriose, especialmente nas mulheres grávidas, nos idosos ou em indivíduos com o sistema imunitário debilitado, de maneira esporádica ou em forma surtos. Realizou-se um estudo observacional, descritivo com o objectivo de se fazer a descrição e a caracterização do surto de listeriose ocorrido na região de Lisboa e Vale do Tejo entre 2009-2011. O período de maior número de casos diagnosticados de listeriose ocorreu entre o mês de Abril e Agosto de 2010. Mas a janela temporal em que ocorreu o surto estendeu-se de Março de 2009 a Janeiro de 2012. Ocorreram 51 casos de internamento com diagnóstico de listeriose, dos quais 25 casos foram confirmados, pela técnica de PFGE, pertencer à mesma estirpe I, sorotipo 4b e pulsotipo 070 e 0101. Na maioria dos casos eram do sexo feminino, com uma média de 57,14 anos de idade e com residência na região de Lisboa e Vale do Tejo. Em 96,08% dos doentes internados com listeriose apresentavam factores de predisposição, comorbilidade e /ou imunossupressão. A bacteriemia foi a manifestação clínica mais frequente, seguida da meningite. A letalidade da listeriose foi de 15,69%.

Utilização de bactérias do ácido láctico como culturas protectoras em enchidos fermentados portugueses

As bactérias do ácido láctico (BAL) são conhecidas pela sua utilização em produtos cárneos fermentados, contribuindo para o desenvolvimento de características sensoriais e melhoria da sua segurança, através de mecanismos naturais com inibição de bactérias deteriorativas e patogénicas. Esta capacidade é chamada de biopreservação, onde a microbiota natural ou a adicionada é usada para aumentar o tempo de prateleira do produto e a sua segurança. O objetivo deste estudo foi avaliar a capacidade de biopreservação de três estirpes de Lactobacillus, previamente isoladas de produtos cárneos fermentados, sendo caracterizado o seu potencial bacteriocinogénico contra diversos microrganismos patogénicos e deteriorativos que podem ser encontrados em produtos cárneos. Duas estirpes foram selecionadas para serem avaliadas em ensaios de antagonismo contra L. innocua, tendo sido inoculadas em massa tradicional de chouriço e em carne sem condimentações, com mimetização de suas fases de fabrico a 7 ºC e 20 ºC. Nesses ensaios a evolução da microbiota deteriorativa e tecnológica foi estudada assim como a capacidade de inibir Listeria sp. de forma a seleccionar as estirpes mais adequadas a participar em culturas protectoras. Face às diferentes condições de estudos pode-se concluir que as três estirpes testadas apresentaram potencial bacteriogénico para com a microbiota deteriorativa e patogénica. Dentre as duas estirpes testadas nos ensaios in vitro, a estirpe L. plantarum P3B7 apresentou resultados inibitórios de aeróbios totais a 30 ºC e Listeria innocua mais desejáveis em ambos os ensaios.

Implementation of membrane processes for improvement of the detection of food contaminants

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities (http://www.em3e.eu)

Ocorrência de Listeria monocytogenes em queijo artesanal tipo coalho comercializado na cidade de Manaus-AM, Brasil

A ocorrência de Listeria sp. em queijo artesanal tipo coalho comercializado na cidade de Manaus foi estudada. No período de março a maio de 1998 foram coletadas 58 amostras do produto em feiras e mercados localizados em seis zonas da cidade de Manaus. Em duas amostras de queijo foram identificadas duas espécies de Listeria: L. monocytogenes e L. innocua. A primeira espécie é reconhecidamente patogênica para o ser humano. Os resultados apresentados demonstraram haver risco de transmissão de enfermidades pelo consumo de queijos processados sem os devidos cuidados de higiene.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Species and serovars of the genus Listeria lsolated from different sources in Brazil from 1971 to 1997

Using phenotype techniques, characterization was made to species and serovar of 3,112 strains of Listeria, isolated from different sources of infection such as human (247-7.9%) and animals (239-7.6%), as well as from various routes of infection, including food (2,330-74.8%) and environmental constituents (296-9.5%), all coming from different regions of the country and collected during the period 1971-1997. The following species were recovered in the cultures analysed: L. monocytogenes (774-24.8%), L. innocua (2,269-72.9%), L. seeligeri (37-1.1%), L. welshimeri (22-0.7%), L. grayi (9-0.2%), and L. ivanovii (1-0.03%). L. monocytogenes was represented by ten serovars, the most prevalent being 4b (352-11.3%), 1/2a (162-5.2%), and 1/2b (148-4.7%). The predominant serovar in L. innocua was 6a (2,093-67.2%). Considerations about laboratory methods for diagnosis and epidemiological aspects are presented on the basis of the results obtained.

Espécies e sorovares de Listeria isolados de animais doentes e portadores no Brasil

A análise fenotípica de 246 amostras do gênero Listeria isolados de animais portadores e doentes, provenientes de três regiões do país, colecionadas no período de 1971 a 2000, permitiu caracterizar as espécies e sorovares prevalentes. Dentre os animais predominaram os espécimes fecais de bovinos normais (217 amostras, 88,2%), em contraposição aos 29 isolados (11,7%) de Listeria de animais doentes, apresentando comprometimento do sistema nervoso central (15 amostras, 6,0%) e outras localizações sistêmicas (14 amostras, 5,6%). Quanto às espécies e sorovares, predominaram L. innocua 6a e não tipável (140 amsotras, 56,9%) e L.monocytogenes 4a (37 amostras, 15,0) e 4b (22 amostras, 8,9%) principalmente nas fezes de bovinos hígidos e nos animais doentes, L. monocytogenes sorovares 4b (14 amostras, 5,6%) com destaque nos ruminantes e 1a (8 amostras, 3,2%) incidindo nas outras espécies animais (roedores e canídeos) e tendo localizações prevalentes em áreas distintas ao sistema nervoso central.