Listeria monocytogenes serotype 1/2b and 4b isolates from human clinical cases and foods show differences in tolerance to refrigeration and salt stress.

Control of Listeria monocytogenes in food processing facilities is a difficult issue because of the ability of this microorganism to form biofilms and adapt to adverse environmental conditions. Survival at high concentrations of sodium chloride and growth at refrigeration temperatures are two other important characteristics of L. monocytogenes isolates. The aim of this study was to compare the growth characteristics under stress conditions at different temperatures of L. monocytogenes serotypes responsible for the majority of clinical cases from different sources. Twenty-two L. monocytogenes isolates, 12 from clinical cases (8 serotype 4b and 4 serotype 1/2a) and 10 from food (6 serotype 4b and 4 serotype 1/2a), and an L. monocytogenes Scott A (serotype 4b) reference strain were analyzed for the ability to grow in brain heart infusion broth plus 1.9 M NaCl (11%) at 4, 10, and 25°C for 73, 42, and 15 days, respectively. The majority of L. monocytogenes strains was viable or even grew at 4°C and under the high osmotic conditions usually used to control pathogens in the food industry. At 10°C, most strains could adapt and grow; however, no significant difference (P > 0.05) was found for lag-phase duration, maximum growth rate, and maximum cell density. At 25°C, all strains were able to grow, and populations increased by up 5 log CFU/ml. Clinical strains had a significantly longer lag phase and lower maximum cell density (P < 0.05) than did food strains. Regarding virulence potential, no significant differences in hemolytic activity were found among serotypes; however, serotype 4b strains were more invasive in Caco-2 cells than were serotype 1/2a strains (P < 0.05). The global tendency of decreasing NaCl concentrations in processed foods for health reasons may facilitate L. monocytogenes survival and growth in these products. Therefore, food companies must consider additional microbial growth barriers to assure product safety.

Contributions of σ(B) and PrfA to Listeria monocytogenes salt stress under food relevant conditions.

Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25°C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25°C in the presence of 1.9M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

Listeria monocytogenes in two different poultry facilities: Manual and automatic evisceration.

Listeriosis is a serious foodborne disease caused by Listeria monocytogenes, a pathogen often found in food processing plants. Poultry meat and its derivatives may harbor L. monocytogenes even if good manufacturing practices are implanted in abattoirs. Little information exists in Brazil on the frequency of L. monocytogenes contamination, even though the country is considered the top poultry meat exporter in the world. This study attempted to compare 2 exporters poultry facilities following same the standards but differing only in manual (plant M) or automatic (plant A) evisceration. Eight hundred fifty-one samples from food, food contact and non-food contact surfaces, water, and workers' hands were collected from cage to finished products over a 1-yr period. In plant A, 20.1% of the samples were positive for L. monocytogenes, whereas in plant M, 16.4% was found. The greatest incidence of contamination with the pathogen in plant A was found in non-food contact surfaces (27.3%), while in plant M, it was found in products (19.4%). The most prevalent serovars were 1/2a or 3a (plant M) and 4b, 4d, or 4e (plant A). Despite having proper hygiene and good manufacturing practices, controlling the entry and persistence of L. monocytogenes in processing facilities remains a formidable task.

Quantitative evaluation of Listeria monocytogenes in fresh and processed surubim fish (Pseudoplatystoma sp) / Avaliação quantitativa de Listeria monocytogenes em surubim (Pseudoplatystoma sp) fresco e processado

L. monocytogenes is a foodborne psychrotrophic bacterial pathogen of special importance for minimally processed foods. In this work, it was enumerated in samples of surubim fish by MPN technique. The population of L. monocytogenes was estimated as < 0.012 MPN/cm² in fresh and < 0.03 MPN/g in minimally processed fish.

L. monocytogenes é um patógeno psicrotrófico transmitido por alimentos, de importância especial para alimentos minimamente processados. Neste trabalho, a bactéria foi enumerada em amostras de peixe surubim utilizando-se a técnica do NMP. A população de L. monocytogenes foi estimada como < 0.012 NMP/cm² do peixe fresco e < 0.03 NMP/g do peixe minimamente processado.

Epidemiological Survey of Listeria monocytogenes in a gravlax salmon processing line / Epidemiologia de Listeria monocytogenes em uma linha de processamento de salmão gravlax

Listeria monocytogenes is a cause of concern to food industries, mainly for those producing ready-to-eat (RTE) products. This microorganism can survive processing steps such as curing and cold smoking and is capable of growing under refrigeration temperatures. Its presence in RTE fish products with extended shelf life may be a risk to the susceptible population. One example of such a product is gravlax salmon; a refrigerated fish product not exposed to listericidal processes and was the subject of this study. In order to evaluate the incidence and dissemination of L. monocytogenes 415 samples were collected at different steps of a gravlax salmon processing line in São Paulo state, Brazil. L. monocytogenes was confirmed in salmon samples (41 percent), food contact surfaces (32 percent), non-food contact surfaces (43 percent) and of food handlers' samples (34 percent), but could not be detected in any ingredient. 179 L. monocytogenes isolates randomly selected were serogrouped and typed by PFGE. Most of L. monocytogenes strains belonged to serogroup 1 (73 percent). 61 combined pulsotypes were found and a dendrogram identified six clusters: most of the strains (120) belonged to cluster A. It was suggested that strains arriving into the plant via raw material could establish themselves in the processing environment contaminating the final product. The wide dissemination of L. monocytogenes in this plant indicates that a great effort has to be taken to eliminate the microorganism from these premises, even though it was not observed multiplication of the microorganism in the final product stored at 4ºC up to 90 days.

Listeria monocytogenes é um patógenode grande preocupação para as indústrias alimentícias, principalmente aquelas produtoras de alimentos prontos para consumo (RTE). Este microrganismo pode sobreviver às etapas de cura e defumação a frio, além de tolerar temperaturas de refrigeração. A presença de L. monocytogenes em pescados RTE com vida de prateleira longa representa um risco para a população susceptível, sendo o salmão gravlax deste tipo de produto. No presente estudo avaliou-se a incidência e disseminação de L. monocytogenes em 415 amostras de salmão gravlax obtidas de diferentes etapas de processamento de uma indústria localizada no Estado de São Paulo. A presença de L. monocytogenes foi confirmada em amostras de salmão (41 por cento), superfícies de contato (32 por cento) e não contato (43 por cento) e manipuladores (34 por cento), porém não se isolou o microrganismo em nenhum ingrediente. Do total de cepas isoladas, 179 destas foram escolhidas aleatoriamente e submetidas a sorologia e tipagem por PFGE. A maioria dos isolados pertenceu ao sorogrupo 1 (73 por cento), sendo identificados 61 pulsotipos quando se combinou os resultados de sorologia e PFGE e 6 clusters foram distribuídos em um dendrograma. O cluster A agrupou a maioria das cepas (120). Pode-se sugerir que as cepas foram introduzidas na linha de processamento por meio da matéria prima e contaminando o produto final. Estes resultados indicam que a eliminação de L. monocytogenes deste estabelecimento requer um grande esforço, ainda que o microrganismo não se multiplicou no produto final estocado a 4ºC por 90 dias.

Epidemiological Survey of Listeria monocytogenes in a gravlax salmon processing line.

Listeria monocytogenes is a cause of concern to food industries, mainly for those producing ready-to-eat (RTE) products. This microorganism can survive processing steps such as curing and cold smoking and is capable of growing under refrigeration temperatures. Its presence in RTE fish products with extended shelf life may be a risk to the susceptible population. One example of such a product is gravlax salmon; a refrigerated fish product not exposed to listericidal processes and was the subject of this study. In order to evaluate the incidence and dissemination of L. monocytogenes 415 samples were collected at different steps of a gravlax salmon processing line in São Paulo state, Brazil. L. monocytogenes was confirmed in salmon samples (41%), food contact surfaces (32%), non-food contact surfaces (43%) and of food handlers' samples (34%), but could not be detected in any ingredient. 179 L. monocytogenes isolates randomly selected were serogrouped and typed by PFGE. Most of L. monocytogenes strains belonged to serogroup 1 (73%). 61 combined pulsotypes were found and a dendrogram identified six clusters: most of the strains (120) belonged to cluster A. It was suggested that strains arriving into the plant via raw material could establish themselves in the processing environment contaminating the final product. The wide dissemination of L. monocytogenes in this plant indicates that a great effort has to be taken to eliminate the microorganism from these premises, even though it was not observed multiplication of the microorganism in the final product stored at 4°C up to 90 days.

Quantitative evaluation of Listeria monocytogenes in fresh and processed surubim fish (Pseudoplatystoma sp).

L. monocytogenes is a foodborne psychrotrophic bacterial pathogen of special importance for minimally processed foods. In this work, it was enumerated in samples of surubim fish by MPN technique. The population of L. monocytogenes was estimated as < 0.012 MPN/cm(2) in fresh and < 0.03 MPN/g in minimally processed fish.

Quantificação de Listeria monocytogenes em salames fatiados embalados a vácuo / Occurrence of Listeria monocytogenes in pre-sliced vacuum-packaged

There is scarce information in Brazil and other South American countries about the occurrence of Listeria monocytogenes in food, mainly refrigerated ready-to-eat products. The consumption of sliced vacuum-packaged meat products has increased in the last few years. Nevertheless, a complete assessment of the risk associated with L. monocytogenes in these products is still necessary. Because of the production and storage characteristics of these products, they can be considered potential vehicles for L. monocytogenes to humans, mainly immunocompromised, elderly, and pregnant women. The objectives of this study was to evaluate the population of L. monocytogenes in salami, a ready-to-eat meat product with extended shelf life, acquired in retail stores in São Paulo-Brazil. The three-tube most probable number technique was used and the methodology was that from Health Canada. Strains were biochemically identified and serotyped. Among the 45 samples, 3 (6.7 per cent) harboured 9.2 MPN/g of L. monocytogenes and the others < 0.3 MPN/g. All the strains belonged to serotypes 1/2a and 1/2b, the most frequent serotypes found in food everywhere. Even being low, the population of L. monocytogenes found in this product could be a cause of concern to public health authorities as it can pose a threat to population at risk. This contamination highlights the importance of implementing systems like HACCP to assure safe products to consumers.

Evaluation of two commercial methods for the detection of Listeria sp. and Listeria monocytogenes in a chicken nugget processing plant.

This study measures the detection performances of two rapid test systems (Listeria Rapid Test Clearview and Bax system) for the screening of Listeria sp. and Listeria monocytogenes, respectively. A total of 413 samples from different sources (product from (i) different stages of processing, (ii) different environments, and (iii) different food handlers), collected from a chicken nugget processing plant, were analysed by both rapid methods and a cultural method consisting of pre-enrichment, enrichment, and isolation onto selective agars (PALCAM, LPM, and HCLA). Overall, results showed an excellent correlation between data obtained using Clearview and the cultural method, with Clearview presenting an efficiency of 99%. Bax showed a lower correlation using the cultural method, with an efficiency of 71.1%. The type of sample did not affect the efficiency of Clearview, which varied from 98.1% for product samples to 100% for environmental and food handler samples, while for Bax it had a marked influence. Efficiency of Bax varied from as high as 100% for food handlers to 37.9% for product samples.

Evaluation of motility enrichment on modified semi-solid Rappaport-Vassiladis medium (MSRV) for the detection of Salmonella in foods.

The detection and identification of Salmonella spp. is still troublesome and time consuming to the food industry. Employing the modified semi-solid Rappaport-Vassiliadis medium (MSRV), presumptive results for Salmonella can be obtained in 48 h, representing an interesting alternative to the standard methods. The specificity and sensitivity of the MSRV method were evaluated in this research. The efficiency of this method was also compared with the methodology recommended by the US Food and Drug Administration (FDA) using bismuth sulfite agar, XLT4 agar and Rambach agar. A total of 146 food samples comprised of 41 chicken thighs, 35 Brazilian fresh pork sausages, 35 samples of cocoa powder and/or granulated cocoa and 35 samples of grated fresh coconut, were examined. Overall, the rapid method (direct + indirect) and the standard culture detected 96.1% and 84.6% of the positive samples, respectively. No Salmonella was detected in the coconut or cocoa samples by any of the methods. Eighteen (43.9%) chicken thigh samples were contaminated with the microorganism. The rapid method (direct + indirect) and the standard culture detected 94.4% and 88.9% of these, respectively. Salmonella was detected in eight (22.8%) fresh pork sausage samples. The MSRV method detected Salmonella in all eight samples, while the standard gave positive results in six (75%). When compared with the standard method, the indirect method showed 86.4% sensitivity and 96.8% specificity, while the direct MSRV showed a sensitivity of 71.4% and specificity of 99.2%. Combined, both MSRV methods showed 95.5% sensitivity and 96.8% specificity. The MSRV medium also reduces the time necessary for the isolation of Salmonella from foods.

Characterization and evaluation of some virulence markers of Listeria monocytogenes strains isolated from Brazilian cheeses using molecular, biochemical and serotyping techniques.

A total of 207 L. monocytogenes strains isolated from different types of cheeses commercialized in the city of Rio de Janeiro, Brazil, were serotyped and evaluated for their ability to produce beta-haemolysin and lecithinase and to adsorb Congo red dye. Of the 207 strains, 59.9, 27.5 and 12.6% belonged to serotypes 1/2a, 1/2b and 4b, respectively. In addition, 175 strains of L. monocytogenes produced lecithinase while strains of the other species did not. Some of the non-L. monocytogenes strains adsorbed the dye Congo red, while some L. monocytogenes did not. Statistical analysis of the results showed significant differences (P < 0.05) amongst the virulence tests and the three serotypes found. In the present study, 32 L. monocytogenes strains were also analyzed by RAPD (randomly amplified polymorphic DNA). RAPD analysis allowed the discrimination among strains of different serotypes, as well as among strains of the same serotype. It is important to emphasize that the use of more than one primer is needed for characterization of L. monocytogenes strains. With RAPD the strains were grouped into six different profiles, some of them common for strains belonging to different serotypes. The results also indicated a close genetic relationship among strains of different serotypes.

Production of mortadella: behavior of Listeria monocytogenes during processing and storage conditions.

The presence of Listeria monocytogenes in ready-to-eat meat products is cause of concern to the food industry as well as to health authorities. Studies were conducted to evaluate the presence of L. monocytogenes in mortadellas acquired at retail stores and to evaluate the fate of two levels of a L. monocytogenes pool spiked in two different formulations of the product, cooked under commercial conditions and stored at refrigeration (7°C) and room temperature (25°C). Among the samples collected at different retail stores, 26.7% harboured L. monocytogenes. Regarding to the fate of L. monocytogenes in mortadella, periodically, samples were taken and the surviving L. monocytogenes cells in the spiked products were counted by the MPN procedure. Populations of <0.35 MPN/g of L. monocytogenes were found in these samples. It could be concluded that the heat treatment was effective to reduce 3-log of L. monocytogenes independent of formulation or storage conditions.

Incidence and significance of Listeria in fish and fish products from Latin America.

The incidence and importance of Listeria spp. in fish and fishery products in Latin American countries is reviewed. There are very few papers dealing with this subject, however it is known that Listeria can be an important problem even for fisheries of tropical countries. The importance of GMP and HACCP implementation is also discussed.

Incidence of Listeria spp. and Salmonella spp. in horsemeat for human consumption.

The incidence of Salmonella spp., Listeria spp. and Listeria monocytogenes in horsemeat for human consumption was investigated. One-hundred and twenty-one samples of frozen horsemeat collected from two Brazilian abattoirs were analysed over a period of 1 year. Twenty-two samples (18.2%) were positive for Listeria spp. with nine (7.4%) containing L. monocytogenes. None of the samples harbored Salmonella spp.

Use of Molecular Typing Methods To Trace the Dissemination of Listeria monocytogenes in a Shrimp Processing Plant.

Volume 62, no. 2, p. 705, column 2, line 5 from bottom: "neutralized with chlorine" should read "chlorine neutralized by the addition of 5 ml of a 1% solution of sodium thiosulfate." Page 706, Table 1, footnote b: Footnote b should read "The designation in parentheses is the area or type of sample collected as indicated in Table 3." Page 709, Tables 3 and 4: Tables 3 and 4 should read as shown below. [This corrects the article on p. 705 in vol. 62.].

Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant.

Molecular typing of bacteria has been widely used in epidemiological studies but not as extensively for tracing the transmission of pathogenic bacteria in food plants. This study was conducted to examine the potential use of two molecular typing methods, random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE), to trace Listeria monocytogenes contamination in a shrimp processing plant. Ribotyping and phase typing were also performed on a select number of strains. One hundred fifteen strains of L. monocytogenes collected in different areas of a shrimp processing plant were first serotyped and then subtyped by molecular typing. RAPD and PFGE showed great promise for typing L. monocytogenes isolates since distinguishable and reproducible DNA polymorphisms were obtained. When the composite profile from both (RAPD and PFGE) methods was generated, there was an increase in the discriminatory power to discern differences between strains of L. monocytogenes. The results indicated that environmental strains all fell into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp isolates belonging to one profile group were found in different areas of the processing line. This same profile group was also present in food handlers from the processing and packaging areas of the plant.

Incidence of Listeria species in raw and pasteurized milk produced in São Paulo, Brazil.

The present study evaluated the incidence of Listeria spp. in raw and pasteurized milk from a Brazilian dairy plant. Ten samples of each type of milk (raw types B and C and pasteurized types B and C) were collected monthly from October 1989 to September 1990 (except in December), comprising 440 samples (110 samples of each type of milk). The recovery of Listeria spp. was carried out using LPM (lithium chloride phenylethanol moxalactam) and MOX (modified Oxford) agars, after a two-step enrichment procedure in Listeria enrichment broth (LEB) and Fraser broth. Overall, 12.7% of raw milk samples, 0.9% of pasteurized milk samples and 6.8% of total of milk samples were positive for Listeria spp., while 9.5% of raw milk samples, none of the pasteurized milk samples and 4.8% of total milk samples, were positive for Listeria monocytogenes. Raw milk also contained L. innocua (9.5%), L. welshimeri (0.9%) and L. grayi (0.4%). Pasteurized milk contained only L. innocua (0.9%).