Dataset Information

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping.

ABSTRACT: BACKGROUND: Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES's Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes. RESULTS: A panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively. CONCLUSIONS: The discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

SUBMITTER: Roussel S

PROVIDER: S-EPMC3561277 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping.

Roussel Sophie S Félix Benjamin B Grant Kathie K Dao Trinh Tam TT Brisabois Anne A Amar Corinne C

BMC microbiology 20130124

<h4>Background</h4>Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES's Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecula ...[more]

PMID: 23347599

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Project description:UnlabelledListeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety.ImportanceThis study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices.

Project description:The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.

Dataset Information

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping.

Publications

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping.

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