The effect of biogenic amine production by single bacterial cultures and metabiosis on cold-smoked salmon

AIM: Biogenic amines are important indicators of spoilage in vacuum-packed cold-smoked salmon. It is the aim of this study to identify bacteria responsible for biogenic amine production in cold-smoked salmon. METHODS AND RESULTS: The present study identified spoilage microflora from cold-smoked salmon and determined biogenic amine production of single and co-cultures growing in cold-smoked salmon. Photobacterium phosphoreum was the only species that produced histamine when inoculated on sterile cold-smoked salmon. Production of putrescine was enhanced 10-15 times when cultures of Serratia liquefaciens or Hafnia alvei were grown with Carnobacterium divergens or Lactobacillus sakei subsp. carnosus. This phenomenon was explained by interspecies microbial metabolism of arginine, i.e., metabiosis. CONCLUSIONS: The amounts of biogenic amines produced by single and co-cultures corresponded to those observed during spoilage of naturally-contaminated cold-smoked salmon. Photobacterium phosphoreum and Lact. curvatus were identified as the specific spoilage organisms in cold-smoked salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the specific spoilage organism is needed before a model can be developed for shelf-life predictions of cold-smoked salmon.     

Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods.

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5 degrees C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10(6) CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5 degrees C under an N(2) atmosphere when mean cell densities increased to 10(6) CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10(6) CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C

AIMS: To evaluate the microbial spoilage, formation of biogenic amines and shelf life of chilled fresh and frozen/thawed salmon packed in a modified atmosphere and stored at 2 degrees C. METHODS AND RESULTS: The dominating microflora, formation of biogenic amines and shelf life were studied in two series of storage trials with naturally contaminated fresh and thawed modified atmosphere-packed (MAP) salmon at 2 degrees C. Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon at more than 10(6) cfu g(-1) and the activity of this specific spoilage organism (SSO) limited the shelf life of the product to ca 14 and 21 d in the two experiments. Despite the high levels of P. phosphoreum, less than 20 mg kg(-1) histamine was observed in fresh MAP salmon prior to sensory spoilage. Freezing eliminated P. phosphoreum and extended the shelf life of MAP salmon at 2 degrees C by 1-2 weeks. Carnobacterium piscicola dominated the spoilage microflora of thawed MAP salmon and probably produced the ca 40 mg kg(-1) tyramine detected in this product at the end of its shelf life. CONCLUSIONS: Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon but produced only small amounts of biogenic amines in this product. The elimination of P. phosphoreum by freezing allowed this bacteria to be identified as the SSO in fresh MAP salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of P. phosphoreum as the SSO in fresh MAP salmon facilitates the development of methods to determine and predict the shelf life of this product, as previously shown with fresh MAP cod.     

Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10⁶ CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N₂ atmosphere when mean cell densities increased to 10⁶ CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10⁶ CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Research of quality indices for cold-smoked salmon using a stepwise multiple regression of microbiological counts and physico-chemical parameters

AIMS: The aim of the study was to assess the relationships between the remaining shelf-life (RSL) of cold-smoked salmon and various microbiological and physico-chemical parameters, using a multivariate data analysis in the form of stepwise forward multiple regression. METHODS AND RESULTS: Thirteen batches of French cold-smoked salmon were analysed weekly during vacuum-packed storage at 5 degrees C for their lipid, water, salt, phenol, pH, total volatile basic nitrogen (TVBN) and trimethylamine contents, total psychrotrophic count, lactic acid bacteria, lactobacilli, B. thermosphacta, Enterobacteriaceae and yeast counts. At the sensory rejection time, the flora was dominated by lactobacilli, lactobacilli/Enterobacteriaceae or Carnobacteria/B. thermosphacta. Shelf-life was very variable (1->6 weeks) and was related to the initial Enterobacteriaceae load (P < 0.05), depending on hygienic conditions in the smokehouse. High correlations existed between the RSL and lactobacilli count (P < 0.01), yeast count (P < 0.05) and TVBN concentration (P < 0.01). A polynomial fitting the RSL as a function of those three factors was proposed (R(2) = 0.80). Assuming that lactobacilli count could not exceed 109 cfu g-1, a minimum of 36 mg-N 100 g-1 was necessary for a product to be rejected, with a yeast count of 104 cfu g-1. CONCLUSION: Estimation of cold-smoked salmon quality is possible by measuring three parameters: lactobacilli and yeast counts and TVBN concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The technical content is important for the smoked salmon industry and for development of quality standards for cold-smoked salmon.     

Biogenic amine formation and microbial spoilage in chilled garfish (Belone belone belone)--effect of modified atmosphere packaging and previous frozen storage

AIMS: To evaluate biogenic amine formation and microbial spoilage in fresh and thawed chilled garfish. METHODS AND RESULTS: Storage trials were carried out with fresh and thawed garfish fillets at 0 or 5 degrees C in air or in modified atmosphere packaging (MAP: 40% CO2 and 60% N2). During storage, sensory, chemical and microbial changes were recorded and histamine formation by isolates from the spoilage microflora was evaluated at 5 degrees C. Photobacterium phosphoreum was responsible for histamine formation (>1000 ppm) in chilled fresh garfish. The use of MAP did not reduce the histamine formation. Strongly histamine-producing P. phosphoreum isolates formed 2080-4490 ppm at 5 degrees C, whereas below 60 ppm was formed by other P. phosphoreum isolates. Frozen storage inactivated P. phosphoreum and consequently reduced histamine formation in thawed garfish at 5 degrees C markedly. CONCLUSIONS: Photobacterium phosphoreum can produce above 1000 ppm of histamine in chilled fresh garfish stored both in air and in MAP. Freezing inactivates P. phosphoreum, extends shelf life and markedly reduces histamine formation in thawed MAP garfish during chilled storage. SIGNIFICANCE AND IMPACT OF THE STUDY: At 5 degrees C, more than 1000 ppm of histamine was formed in garfish; thus even when it is chilled this product represents a histamine fish-poisoning risk.     

Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon

Aims: To evaluate and model the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon. Methods and Results: Growth kinetics of L. monocytogenes, lactic acid bacteria (LAB), Enterobacteriaceae, enterococci and Photobacterium phosphoreum were determined in two series of challenge tests with sliced and vacuum-packed cold-smoked salmon (SVP-CSS). The product contained a high level of smoke components and at 2°C levels of L. monocytogenes increased <100-fold in 193 days. Without the addition of spoilage micro-organisms, L. monocytogenes reached ca 10⁸ CFU g⁻¹ at 5, 10, 17·5 and 25°C. Inoculation with spoilage micro-organisms reduced this level to 10²–10⁴ CFU g⁻¹. LAB dominated the spoilage microfora of SVP-CSS and competition between LAB and L. monocytogenes in SVP-CSS was appropriately described by a simple expansion of the Logistic model. This interaction model aided in predicting the growth of L. monocytogenes in naturally contaminated SVP-CSS when it was used in combination with expanded versions of existing secondary models for L. monocytogenes and LAB. Conclusions: Temperature, water activity/NaCl, simultaneous growth of LAB, smoke components and to a lesser extent lactate and pH control growth of L. monocytogenes in SVP-CSS. These factors must be included in mathematical models to predict growth of the pathogen in this product. Significance and Impact of the Study: The suggested predictive model can be used to support assessment and management of the human health risk due to L. monocytogenes in SVP-CSS.     

Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham

In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum-packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non-identifiable group of leuconostoc-like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram-negative at the beginning of processing to highly Gram-positive prior to cooking was noted. Listeria monocytogenes cross-contaminated ham during tumbling. However, the pathogen was always absent from the vacuum-packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.     

Effects of high-pressure processing on Listeria monocytogenes, spoilage microflora and multiple compound quality indices in chilled cold-smoked salmon

AIMS: To evaluate the effect of high-pressure processing (HPP) on Listeria monocytogenes, microbial and chemical changes and shelf-life in chilled cold-smoked salmon (CSS). METHODS AND RESULTS: First, challenge tests with L. monocytogenes were carried out using HPP of the product at 0.1 (control), 150, 200 and 250 MPa. Secondly, storage trials with the naturally contaminated product and HPP at 0.1 (control) and 200 MPa were realized. Shelf-life, microbial changes and chemical changes were determined and existing predictive models and multiple compound quality indices evaluated. HPP with 250 MPa did not inactivate L. monocytogenes but significant lag phases of 17 and 10 days were observed at ca 5 and 10 degrees C, respectively. HPP with 200 MPa had a marked effect on both colour and texture of CSS. CONCLUSIONS: High-pressure processing was unable to prevent growth of L. monocytogenes or spoilage of chilled CSS. Existing mathematical models allowed growth rates of L. monocytogenes and shelf-life of samples without high-pressure treatments to be predicted. SIGNIFICANCE AND IMPACT OF THE STUDY: High-pressure processing seems more appropriate for new types of salmon products than for a classical product like CSS where consumers expect specific quality attributes.     

rpoB-PCR amplified gene and temporal temperature gradient gel electrophoresis: a rapid tool to analyse bacterial strains representative of cold-smoked salmon microflora

AIM: To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. METHODS AND RESULTS: DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. CONCLUSION: The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis.     

Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.     

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).     

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.     

Identification of lactic acid bacteria from spoilage associations of cooked and brined shrimps stored under modified atmosphere between 0 degrees C and 25 degrees C

AIMS: To evaluate spoilage and identify lactic acid bacteria (LAB) from spoilage associations of cooked and brined shrimps stored under modified atmosphere packaging (MAP) at 0, 5, 8, 15 and 25 degrees C. METHODS AND RESULTS: Bacterial isolates (102) from spoilage associations of cooked and brined MAP shrimps were characterized by phenotypic tests and identified as lactic acid bacteria (78 isolates), other Gram-positive bacteria (13 isolates) and Gram-negative bacteria (11 isolates). A selection of 48 LAB isolates were further characterized and identified by phenotypic tests and SDS-PAGE electrophoresis of whole cell proteins. Selected clusters of LAB isolates were analysed by plasmid profiling, pulsed field gel electrophoresis and 16S rRNA gene sequencing. Enterococcus faecalis was identified in spoilage associations at 15 degrees C and 25 degrees C, and its metabolic activity corresponded to chemical changes in spoiled products. Carnobacterium divergens, a non-motile Carnobacterium sp. nov. and Lactobacillus curvatus were the LAB species observed in spoilage associations of products stored at 0 degrees C, 5 degrees C and 8 degrees C. CONCLUSIONS: Enterococcus spp. and Carnobacterium spp. were the dominant parts of spoilage associations of cooked and brined MAP shrimps stored at high and low temperatures, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The SDS-PAGE technique and simple biochemical keys allowed the majority of LAB isolates from spoilage associations of cooked and brined MAP shrimps to be identified at the species level.     

Sensory, chemical and bacteriological changes during storage of iced squid (Todaropsis eblanae)

AIMS: To relate sensory shelf-life of iced whole and gutted squid to bacterial growth and chemical changes. METHODS AND RESULTS: Cooked mantles from whole and gutted individuals were rejected after 10 and 12 days of storage, respectively, due to ammoniacal off-odours. Rate of production of both ammonia and trimethylamine was highest in the whole lot. Agmatine, which was only present in trace amounts in freshly-caught squid, increased rapidly in both lots. The main microflora at the time of sensory rejection of iced whole squid included Gram-negative, motile and non-fermentative rods, which were psychrophilic and had a requirement for NaCl. 16S rDNA sequence analyses identified the strains as belonging to the genus Pseudoalteromonas. Shewanella putrefaciens, Pseudoalteromonas sp. and Pseudomonas sp. dominated in spoiled gutted squid. Identification of strains from the stomach and digestive gland of recently-captured squid showed that the main flora consisted of Photobacterium phosphoreum. CONCLUSIONS: Spoilage of iced squid is likely to result from a combination of autolytic and bacterial changes. Agmatine seems to be an excellent freshness indicator. Photobacterium phosphoreum may contribute to spoilage through activity in the digestive gland, followed by diffusion of volatile compounds and amines to the mantle. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the psychrophilic nature of P. phosphoreum and Pseudoalteromonas sp., spread-plating and low temperature incubation are recommended for bacteriological evaluation of iced squid.     

Changes in the Microflora of Vacuum-packaged, Irradiated Petrale Sole (Eopsetta jordani) Fillets Stored at 0.5 C

The microfloral changes of irradiated petrale sole fillets during anaerobic (vacuum-packed in cans) refrigerated storage was determined by the identification of 1,260 microbial isolates to the generic level. The samples were irradiated at 0.0, 0.1, 0.2, 0.3, and 0.4 Mrad from a cobalt-60 gamma source, stored at 0.5 C, and examined periodically for spoilage and total microbial population and composition. The preirradiation flora consisted of Achromobacter, Micrococcus, Pseudomonas, coryneforms, Lactobacillus, and Flavobacterium. Immediately after irradiation, Micrococcus, Achromobacter, coryneforms, and Bacillus were predominant. After storage under vacuum, the spoilage flora of the nonirradiated petrale sole was predominantly Pseudomonas; the spoilage flora of the 0.1-Mrad irradiated samples consisted of Pseudomonas and Lactobacillus; and that of the 0.2-, 0.3-, and 0.4-Mrad samples was almost entirely Lactobacillus.     

Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue

Aerobic and anaerobic plate counts were compared for routine monitoring of the microflora, dominated by lactic acid bacteria, developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage. No statistical differences were observed between aerobic and anaerobic enumerations, made on plate count and blood agar plates, of the microflora developing on beef striploins packaged under vacuum or carbon dioxide during 14 weeks' storage at 0°C. With both techniques the spoilage microflora development differed between the two packaging regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced by anaerobic plate counts in the routine microbiological examination of the spoilage microflora developing on chilled meats packaged under anoxic modified atmospheres.     

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.     

Abattoir sources of psychrophilic clostridia causing blown pack spoilage of vacuum-packed chilled meats determined by culture-based and molecular detection procedures

AIMS: To identify the abattoir source(s) of psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Molecular procedures were used to detect the presence of specific 16S rRNA gene fragments of blown pack-causing clostridia in samples collected from a commercial abattoir and its environs. Blown pack-causing clostridia were consistently detected in hide, soil and faecal samples, as well as in samples collected at slaughter plant locations associated with handling of animals and animal carcasses prior to pelt removal. CONCLUSIONS: The data indicate that pelts per se or soil particles/faecal material attached thereto are the most probable primary reservoir of blown pack clostridia in the abattoir. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling blown pack spoilage in commercial meat-processing plants.