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Applicability of biological time temperature integrators as quality and safety indicators for meat products.

https://arctichealth.org/en/permalink/ahliterature146076
Source
Int J Food Microbiol. 2010 Mar 31;138(1-2):119-29
Publication Type
Article
Date
Mar-31-2010
Author
M. Ellouze
J-C Augustin
Author Affiliation
CRYOLOG SA Département R&D. 58, Nantes, France. mellouze@vet-alfort.fr
Source
Int J Food Microbiol. 2010 Mar 31;138(1-2):119-29
Date
Mar-31-2010
Language
English
Publication Type
Article
Keywords
Animals
Colony Count, Microbial
Consumer Product Safety
Food Contamination - analysis - prevention & control
Food Microbiology
Food Packaging - methods
Food Preservation - methods
Humans
Listeria monocytogenes - growth & development
Meat Products - microbiology - standards
Oxygen - metabolism
Poultry Products - microbiology - standards
Salmonella - growth & development
Staphylococcus aureus - growth & development
Temperature
Time Factors
Abstract
The objective of this study was to evaluate (eO), a biological time temperature integrator (TTI) as a quality and safety indicator for ground beef packed under modified atmosphere and spiced cooked chicken slices packed under modified atmosphere. Storage trials and challenge tests were thus performed on several batches of the studied food to monitor and model the behavior of Listeria monocytogenes, Salmonella, Staphylococcus aureus and the indigenous food flora. Then, two different prototypes of the TTI (eO) were set and manufactured according to the studied products shelf lives. The TTI evolution with time at static and dynamic temperatures was monitored and modeled. Finally, exposure assessment models were set and used under several realistic storage profiles to assess the distributions of the concentration of the indigenous food flora and the distributions of the increase in the pathogens populations obtained at the end of the product shelf life or at the end point of the TTI, taking into account the TTIs batch variability. Results showed that in case of poor storage conditions, TTI can reduce the consumer exposure to altered or hazardous foods.
PubMed ID
20074826 View in PubMed
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Cold growth behaviour and genetic comparison of Canadian and Swiss Listeria monocytogenes strains associated with the food supply chain and human listeriosis cases.

https://arctichealth.org/en/permalink/ahliterature256814
Source
Food Microbiol. 2014 Jun;40:81-7
Publication Type
Article
Date
Jun-2014
Author
Carolina Arguedas-Villa
Jovana Kovacevic
Kevin J Allen
Roger Stephan
Taurai Tasara
Author Affiliation
Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Switzerland.
Source
Food Microbiol. 2014 Jun;40:81-7
Date
Jun-2014
Language
English
Publication Type
Article
Keywords
Adaptation, Physiological
Bacterial Proteins - genetics
Canada
Food chain
Food Microbiology
Food Supply
Humans
Listeria monocytogenes - classification - genetics - growth & development - isolation & purification
Listeriosis - microbiology
Phylogeny
Switzerland
Temperature
Abstract
Sixty-two strains of Listeria monocytogenes isolated in Canada and Switzerland were investigated. Comparison based on molecular genotypes confirmed that strains in these two countries are genetically diverse. Interestingly strains from both countries displayed similar range of cold growth phenotypic profiles. Based on cold growth lag phase duration periods displayed in BHI at 4??C, the strains were similarly divided into groups of fast, intermediate and slow cold adaptors. Overall Swiss strains had faster exponential cold growth rates compared to Canadian strains. However gene expression analysis revealed no significant differences between fast and slow cold adapting strains in the ability to induce nine cold adaptation genes (lmo0501, cspA, cspD, gbuA, lmo0688, pgpH, sigB, sigH and sigL) in response to cold stress exposure. Neither was the presence of Stress survival islet 1 (SSI-1) analysed by PCR associated with enhanced cold adaptation. Phylogeny based on the sigL gene subdivided strains from these two countries into two major and one minor cluster. Fast cold adaptors were more frequently in one of the major clusters (cluster A), whereas slow cold adaptors were mainly in the other (cluster B). Genetic differences between these two major clusters are associated with various amino acid substitutions in the predicted SigL proteins. Compared to the EGDe type strain and most slow cold adaptors, most fast cold adaptors exhibited five identical amino acid substitutions (M90L, S203A/S203T, S304N, S315N, and I383T) in their SigL proteins. We hypothesize that these amino acid changes might be associated with SigL protein structural and functional changes that may promote differences in cold growth behaviour between L.?monocytogenes strains.
PubMed ID
24549201 View in PubMed
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Control of bacterial pathogens during processing of cold-smoked and dried salmon strips.

https://arctichealth.org/en/permalink/ahliterature3437
Source
J Food Prot. 2004 Feb;67(2):347-51
Publication Type
Article
Date
Feb-2004
Author
M W Eklund
M E Peterson
F T Poysky
R N Paranjpye
G A Pelroy
Author Affiliation
Mel Eklund & Associates, Inc., 18727 35th N.E., Seattle, Washington 98155, USA.
Source
J Food Prot. 2004 Feb;67(2):347-51
Date
Feb-2004
Language
English
Publication Type
Article
Keywords
Alaska
Animals
Cold
Colony Count, Microbial
Food Contamination
Food Handling - methods - standards
Food Microbiology
Food-Processing Industry - standards
Kinetics
Listeria monocytogenes - growth & development
Research Support, Non-U.S. Gov't
Salmon - microbiology
Smoke
Staphylococcus aureus - growth & development
Temperature
Abstract
Microbiological and chemical changes were determined during the smoking and drying of salmon strips processed at 29 to 31 degrees C for 4 days at a facility in Alaska in 1993. During the process, Staphylococcus aureus populations increased to more than 10(5) CFU/g after 2 to 3 days of processing. Subsequent laboratory studies showed that a pellicle (dried skinlike surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in or under the pellicle were able to grow even when heavy smoke deposition occurred. Under these conditions, an inoculum of 26 CFU/g of S. aureus increased to 10(5) CFU/g after 3 days of processing. Elimination of preprocess drying and reduction in air flow during smoking resulted in smoke deposition before pellicle formation and enabled the product to reach levels of water-phase salt and water activity that inhibit the growth of S. aureus and Listeria monocytogenes. In 1994, these modifications were then applied during processing at an Alaskan facility, and S. aureus could not be detected in the finished product. L. monocytogenes was detected in the raw product area, on the processing tables, and on the raw salmon strips, but it was not detected in the finished product when the smoke was applied before pellicle formation.
PubMed ID
14968968 View in PubMed
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Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis.

https://arctichealth.org/en/permalink/ahliterature179719
Source
Clin Microbiol Infect. 2004 Jun;10(6):562-8
Publication Type
Article
Date
Jun-2004
Author
S. Lukinmaa
K. Aarnisalo
M-L Suihko
A. Siitonen
Author Affiliation
Laboratory of Enteric Pathogens, National Public Health Institute, Helsinki, Finland.
Source
Clin Microbiol Infect. 2004 Jun;10(6):562-8
Date
Jun-2004
Language
English
Publication Type
Article
Keywords
Animals
Automation
Bacterial Typing Techniques
Electrophoresis, Gel, Pulsed-Field
Finland - epidemiology
Food Microbiology
Humans
Listeria monocytogenes - classification - genetics - isolation & purification
Listeriosis - microbiology
Ribotyping
Serotyping
Abstract
Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype 1/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE.
PubMed ID
15191386 View in PubMed
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Effect of potassium lactate and a potassium lactate-sodium diacetate blend on Listeria monocytogenes growth in modified atmosphere packaged sliced ham.

https://arctichealth.org/en/permalink/ahliterature160564
Source
J Food Prot. 2007 Oct;70(10):2297-305
Publication Type
Article
Date
Oct-2007
Author
L A Mellefont
T. Ross
Author Affiliation
Australian Food Safety Centre of Excellence, Tasmanian Institute of Agricultural Research, School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart 7001, Tasmania, Australia. lyndal.mellefont@utas.edu.au
Source
J Food Prot. 2007 Oct;70(10):2297-305
Date
Oct-2007
Language
English
Publication Type
Article
Keywords
Animals
Colony Count, Microbial
Food contamination - analysis
Food Handling - methods
Food Microbiology
Food Packaging - methods
Food Preservation - methods
Food Preservatives - pharmacology
Humans
Lactates - pharmacology
Listeria monocytogenes - drug effects - growth & development
Meat Products - microbiology
Salmonella Food Poisoning - epidemiology - prevention & control
Sodium Acetate - pharmacology
Swine
Temperature
Time Factors
Abstract
Two commercially available organic acid salts, potassium lactate (PURASAL HiPure P) and a potassium lactate-sodium diacetate blend (PURASAL Opti. Form PD 4), were assessed as potential inhibitors of Listeria monocytogenes growth in modified atmosphere packaged (MAP) sliced ham in challenge studies. The influence of the initial inoculation level of L. monocytogenes (10(1) or 10(3) CFU g(-1)) and storage temperature (4 or 8 degrees C) was also examined. The addition of either organic acid salt to MAP sliced ham strongly inhibited the growth of L. monocytogenes during the normal shelf life of the product under ideal refrigeration conditions (4 degrees C) and even under abusive temperature conditions (i.e., 8 degrees C). During the challenge studies and in the absence of either organic acid salt, L. monocytogenes numbers increased by 1000-fold after 20 days at 8 degrees C and 10-fold after 42 days at 4 degrees C. Both organic acid salt treatments were found to be listeriostatic rather than listericidal. The addition of either organic acid salt to the MAP ham also reduced the growth of indigenous microflora, i.e., aerobic microflora and lactic acid bacteria. The influence of these compounds on the risk of listeriosis in relation to product shelf life is discussed.
PubMed ID
17969611 View in PubMed
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Efficacy of neutral electrolyzed water (NEW) for reducing microbial contamination on minimally-processed vegetables.

https://arctichealth.org/en/permalink/ahliterature159055
Source
Int J Food Microbiol. 2008 Mar 31;123(1-2):151-8
Publication Type
Article
Date
Mar-31-2008
Author
Maribel Abadias
Josep Usall
Márcia Oliveira
Isabel Alegre
Inmaculada Viñas
Author Affiliation
IRTA, Centre UdL-IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain. isabel.abadias@irta.cat
Source
Int J Food Microbiol. 2008 Mar 31;123(1-2):151-8
Date
Mar-31-2008
Language
English
Publication Type
Article
Keywords
Colony Count, Microbial
Consumer Product Safety
Disinfectants - pharmacology
Dose-Response Relationship, Drug
Escherichia coli O157 - drug effects - growth & development
Food Contamination - analysis - prevention & control
Food Handling - methods
Food Microbiology
Humans
Hydrogen Peroxide - pharmacology
Lettuce - microbiology
Listeria monocytogenes - drug effects - growth & development
Pectobacterium carotovorum - drug effects - growth & development
Salmonella - drug effects - growth & development
Temperature
Time Factors
Vegetables - microbiology
Abstract
Consumption of minimally-processed, or fresh-cut, fruit and vegetables has rapidly increased in recent years, but there have also been several reported outbreaks associated with the consumption of these products. Sodium hypochlorite is currently the most widespread disinfectant used by fresh-cut industries. Neutral electrolyzed water (NEW) is a novel disinfection system that could represent an alternative to sodium hypochlorite. The aim of the study was to determine whether NEW could replace sodium hypochlorite in the fresh-cut produce industry. The effects of NEW, applied in different concentrations, at different treatment temperatures and for different times, in the reduction of the foodborne pathogens Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 and against the spoilage bacterium Erwinia carotovora were tested in lettuce. Lettuce was artificially inoculated by dipping it in a suspension of the studied pathogens at 10(8), 10(7) or 10(5) cfu ml(-1), depending on the assay. The NEW treatment was always compared with washing with deionized water and with a standard hypochlorite treatment. The effect of inoculum size was also studied. Finally, the effect of NEW on the indigenous microbiota of different packaged fresh-cut products was also determined. The bactericidal activity of diluted NEW (containing approximately 50 ppm of free chlorine, pH 8.60) against E. coli O157:H7, Salmonella, L. innocua and E. carotovora on lettuce was similar to that of chlorinated water (120 ppm of free chlorine) with reductions of 1-2 log units. There were generally no significant differences when treating lettuce with NEW for 1 and 3 min. Neither inoculation dose (10(7) or 10(5) cfu ml(-1)) influenced the bacterial reduction achieved. Treating fresh-cut lettuce, carrot, endive, corn salad and 'Four seasons' salad with NEW 1:5 (containing about 50 ppm of free chlorine) was equally effective as applying chlorinated water at 120 ppm. Microbial reduction depended on the vegetable tested: NEW and sodium hypochlorite treatments were more effective on carrot and endive than on iceberg lettuce, 'Four seasons' salad and corn salad. The reductions of indigenous microbiota were smaller than those obtained with the artificially inoculated bacteria tested (0.5-1.2 log reduction). NEW seems to be a promising disinfection method as it would allow to reduce the amount of free chlorine used for the disinfection of fresh-cut produce by the food industry, as the same microbial reduction as sodium hypochlorite is obtained. This would constitute a safer, 'in situ', and easier to handle way of ensuring food safety.
PubMed ID
18237810 View in PubMed
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Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25 degrees C in air or vacuum packages: in situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.

https://arctichealth.org/en/permalink/ahliterature151703
Source
J Food Prot. 2009 Mar;72(3):531-8
Publication Type
Article
Date
Mar-2009
Author
Eleni Giannou
Athanasia Kakouri
Bojana Bogovic Matijasic
Irena Rogelj
John Samelis
Author Affiliation
National Agricultural Research Foundation, Dairy Research Institute, Katsikas, 45221 Ioannina, Greece.
Source
J Food Prot. 2009 Mar;72(3):531-8
Date
Mar-2009
Language
English
Publication Type
Article
Keywords
Bacteriocins - isolation & purification
Cheese - microbiology
Colony Count, Microbial
Consumer Product Safety
Enterococcus faecium - metabolism
Food Microbiology
Food Packaging - methods
Food Preservation - methods
Humans
Listeria monocytogenes - drug effects - growth & development
Oxygen - metabolism
Polymerase Chain Reaction - methods
Risk assessment
Temperature
Time Factors
Vacuum
Abstract
The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P
PubMed ID
19343941 View in PubMed
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Fate of Listeria monocytogenes , Pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp(+) in Ready-to-Eat Salad during Cold Storage: What Is the Risk to Consumers?

https://arctichealth.org/en/permalink/ahliterature283185
Source
J Food Prot. 2017 Feb;80(2):204-212
Publication Type
Article
Date
Feb-2017
Author
Karin Söderqvist
Susanne Thisted Lambertz
Ivar Vågsholm
Lise-Lotte Fernström
Beatrix Alsanius
Lars Mogren
Sofia Boqvist
Source
J Food Prot. 2017 Feb;80(2):204-212
Date
Feb-2017
Language
English
Publication Type
Article
Keywords
Cold Temperature
Colony Count, Microbial
Escherichia coli O157
Food Microbiology
Humans
Listeria monocytogenes
Sweden
Temperature
Yersinia enterocolitica
Abstract
In this study, we investigated the fate of Listeria monocytogenes , pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp(+) inoculated in low numbers into ready-to-eat baby spinach and mixed-ingredient salad (baby spinach with chicken meat). Samples were stored at recommended maximum refrigerator temperature (8°C in Sweden) or at an abuse temperature (15°C) for up to 7 days. Mixed-ingredient salad supported considerable growth when stored at 15°C during shelf life (3 days), with populations of L. monocytogenes , pathogenic Y. enterocolitica , and E. coli O157:H7 gfp(+) increasing from less than 2.0 log CFU/g on day 0 to 7.0, 4.0, and 5.6 log CFU/g, respectively. However, when mixed-ingredient salad was stored at 8°C during shelf life, only L. monocytogenes increased significantly, reaching 3.0 log CFU/g within 3 days. In plain baby spinach, only pathogenic Y. enterocolitica populations increased significantly during storage for 7 days, and this was exclusively at an abuse temperature (15°C). Thus, mixing ready-to-eat leafy vegetables with chicken meat strongly influenced levels of inoculated strains during storage. To explore the food safety implications of these findings, bacterial numbers were translated into risks of infection by modeling. The risk of listeriosis (measured as probability of infection) was 16 times higher when consuming a mixed-ingredient salad stored at 8°C at the end of shelf life, or 200,000 times higher when stored at 15°C, compared with when consuming it on the day of inoculation. This indicates that efforts should focus on preventing temperature abuse during storage to mitigate the risk of listeriosis. The storage conditions recommended for mixed-ingredient salads in Sweden (maximum 8°C for 3 days) did not prevent growth of L. monocytogenes in baby spinach mixed with chicken meat. Manufacturers preparing these salads should be aware of this, and recommended storage temperature should be revised downwards to reduce the risk of foodborne disease.
PubMed ID
28221975 View in PubMed
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Genome sequencing identifies two nearly unchanged strains of persistent Listeria monocytogenes isolated at two different fish processing plants sampled 6 years apart.

https://arctichealth.org/en/permalink/ahliterature261001
Source
Appl Environ Microbiol. 2013 May;79(9):2944-51
Publication Type
Article
Date
May-2013
Author
Anne Holch
Kristen Webb
Oksana Lukjancenko
David Ussery
Benjamin M Rosenthal
Lone Gram
Source
Appl Environ Microbiol. 2013 May;79(9):2944-51
Date
May-2013
Language
English
Publication Type
Article
Keywords
Animals
Bacterial Proteins - genetics
Bacterial Typing Techniques
Base Sequence
DNA, Bacterial - chemistry - genetics - isolation & purification
Denmark
Food Microbiology
Food Preservation
Food-Processing Industry
Genome, Bacterial - genetics
High-Throughput Nucleotide Sequencing
Humans
Listeria monocytogenes - classification - genetics - isolation & purification
Listeriosis - microbiology
Molecular Sequence Data
Multilocus Sequence Typing
Polymorphism, Single Nucleotide
Random Amplified Polymorphic DNA Technique
Salmon - microbiology
Seafood - microbiology
Sequence Analysis, DNA
Time Factors
Abstract
Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3'-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.
Notes
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PubMed ID
23435887 View in PubMed
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Listeria monocytogenes risk assessment on cold smoked and salt-cured fishery products in Finland - A repeated exposure model.

https://arctichealth.org/en/permalink/ahliterature302857
Source
Int J Food Microbiol. 2019 Sep 02; 304:97-105
Publication Type
Journal Article
Date
Sep-02-2019
Author
Petra Pasonen
Jukka Ranta
Heli Tapanainen
Liisa Valsta
Pirkko Tuominen
Author Affiliation
Finnish Food Authority, Mustialankatu 3, 00790 Helsinki, Finland. Electronic address: petra.pasonen@ruokavirasto.fi.
Source
Int J Food Microbiol. 2019 Sep 02; 304:97-105
Date
Sep-02-2019
Language
English
Publication Type
Journal Article
Keywords
Aged
Animals
Bayes Theorem
Consumer Product Safety
Finland - epidemiology
Fisheries
Food Handling - methods
Foodborne Diseases - epidemiology - microbiology
Humans
Listeria monocytogenes - growth & development
Listeriosis - epidemiology
Risk assessment
Salmon - microbiology
Seafood - microbiology
Smoke
Sodium Chloride
Temperature
Abstract
Listeria monocytogenes causes severe consequences especially for persons belonging to risk groups. Finland is among the countries with highest number of listeriosis cases in the European Union. Although most reported cases appear to be sporadic and the maximum bacterial concentration of 100?cfu/g is not usually exceeded at retail, cold smoked and salt-cured fish products have been noted as those products with great risk especially for the elderly. In order to investigate the listeriosis risk more carefully, an exposure assessment was developed, and laboratory results for cold smoked and salt-cured salmon products were exploited. L. monocytogenes exposure was modeled for consumers in two age groups, the elderly population as a risk group and the working-age population as a reference. Incidence was assessed by estimating bacterial growth in the food products at three temperatures. Bayesian estimation of the risk was based on bacterial occurrence and product consumption data and epidemiological population data. The model builds on a two-state Markov chain describing repeated consumption on consecutive days. The cumulative exposure is probabilistically governed by the daily decreasing likelihood of continued consumption and the increasing bacterial concentrations due to growth. The population risk was then predicted with a Poisson distribution accounting for the daily probabilities of purchasing a contaminated product and the cumulative total probability of infection from its use. According to the model presented in this article, elderly Finns are at a greater risk of acquiring listeriosis than healthy adults. The risk for the elderly does not fully diminish even if the products have been stored at the recommended temperature (between 0 and 3?°C). It can be concluded that the stage after retail, i.e. food handling and storage by consumer or professional kitchens, is essential to protection against listeriosis. The estimation model provides means for assessing the joint impacts of these effects.
PubMed ID
31176965 View in PubMed
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24 records – page 1 of 3.