Pulsed-field gel electrophoresis proficiency testing trials: toward European harmonization of the typing of food and clinical strains of Listeria monocytogenes.

The European Union Reference Laboratory for Listeria monocytogenes (EURL for Lm) coordinates a European network of 35 National Reference Laboratories (NRLs), most of which perform food, environmental, and veterinary Lm strain surveillance in their respective countries. The EURL activities resulted in the recent creation of a database (EURL Lm DB). Typing and related epidemiological data submitted to the EURL Lm DB will be collected and shared by all the NRLs. For a given NRL, the only criterion required in order to submit pulsed-field gel electrophoresis (PFGE) profiles to the database was the successful participation with at least one EURL PFGE and PFGE profile interpretation Proficiency Testing (PT) trial. In this context, the EURL organized a PT trial in 2012 to evaluate the NRL's ability to perform PFGE and profile interpretation. A total of 18 NRLs took part in this study. Upon request from the Food- and Waterborne Diseases and Zoonoses Programme of the European Centre for Disease Prevention and Control, 10 National Public Health Reference Laboratories (NPHLs) also took part in this PT trial. Of the 28 participating laboratories, 16 obtained results classified as "good" or "satisfactory." These 16 laboratories included 10 NRLs (56%) and 6 NPHLs (60%). Of the 22 NRLs and NHPLs that participated in the part of the PT trial related to PFGE profile interpretation, 11 laboratories obtained good results. These 11 laboratories included eight NRLs, which therefore can now submit profiles to the EURL Lm DB. This PT trial provided a valuable opportunity to facilitate and to stimulate the sharing of reproducible PFGE profiles between human and food reference laboratories.

Pulsed-Field Gel Electrophoresis Proficiency Testing Trials: Toward European Harmonization of the Typing of Food and Clinical Strains of Listeria monocytogenes.

Abstract The European Union Reference Laboratory for Listeria monocytogenes (EURL for Lm) coordinates a European network of 35 National Reference Laboratories (NRLs), most of which perform food, environmental, and veterinary Lm strain surveillance in their respective countries. The EURL activities resulted in the recent creation of a database (EURL Lm DB). Typing and related epidemiological data submitted to the EURL Lm DB will be collected and shared by all the NRLs. For a given NRL, the only criterion required in order to submit pulsed-field gel electrophoresis (PFGE) profiles to the database was the successful participation with at least one EURL PFGE and PFGE profile interpretation Proficiency Testing (PT) trial. In this context, the EURL organized a PT trial in 2012 to evaluate the NRL's ability to perform PFGE and profile interpretation. A total of 18 NRLs took part in this study. Upon request from the Food- and Waterborne Diseases and Zoonoses Programme of the European Centre for Disease Prevention and Control, 10 National Public Health Reference Laboratories (NPHLs) also took part in this PT trial. Of the 28 participating laboratories, 16 obtained results classified as "good" or "satisfactory." These 16 laboratories included 10 NRLs (56%) and 6 NPHLs (60%). Of the 22 NRLs and NHPLs that participated in the part of the PT trial related to PFGE profile interpretation, 11 laboratories obtained good results. These 11 laboratories included eight NRLs, which therefore can now submit profiles to the EURL Lm DB. This PT trial provided a valuable opportunity to facilitate and to stimulate the sharing of reproducible PFGE profiles between human and food reference laboratories.

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

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.

Pulsed-field gel electrophoresis, conventional, and molecular serotyping of Listeria monocytogenes from food proficiency testing trials toward an harmonization of subtyping at European level.

The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.

Antimicrobial resistance of Listeria monocytogenes isolates from food and the environment in France over a 10-year period.

In order to assess antimicrobial resistance in Listeria monocytogenes, 202 food and environmental isolates from 1996 to 2006 were tested. Only four strains displayed acquired resistance. Resistance to erythromycin, tetracycline-minocycline, and trimethoprim was evidenced, and the genes erm(B), tet(M), and dfrD, already found in L. monocytogenes, were detected.

Semi-automated repetitive-sequence-based polymerase chain reaction compared to pulsed-field gel electrophoresis for Listeria monocytogenes subtyping.

Listeriosis is a severe infection that mainly affects pregnant women, neonates, and immuno-compromised adults. The commercially available semi-automated repetitive-sequence-based polymerase chain reaction assay system, DiversiLab, has been successfully used for subtyping several species of bacteria. In this article we compare the DiversiLab System with macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), which is currently the gold standard for molecular subtyping of Listeria monocytogenes. We used a panel of 116 human and food L. monocytogenes isolates for the comparative evaluation. Among these isolates, there were 4 pairs of duplicates, 13 strains were epidemiologically related, and the remaining food isolates were epidemiologically unrelated. The isolates of different serotypes represented distinct DiversiLab types (DTs) and ApaI/AscI-PFGE types except for one DT-containing isolates of two serotypes, 4b and 1/2b. The four duplicates displayed the same DT and ApaI/AscI PFGE type demonstrating the good reproducibility of the two methods. The epidemiologically related strains were clustered in the same DT and PFGE type. The Simpson's index of diversity was 0.954; 0.988; 0.994; and 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE, and AscI/ApaI-PFGE, respectively. Thus, PFGE was more discriminating than DiversiLab. However, for 1/2a serotype strains, six AscI-PFGE, three ApaI-PFGE, and one ApaI/AscI PFGE type were divided into different DTs. DiversiLab enabled a good discrimination between serotype 1/2a strains. DiversiLab is less labor intensive than PFGE and provides results in <24 hours compared with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria has been obtained. On the basis of these results, DiversiLab may be useful for tracking the source of contamination in food-processing facilities and their environments. Also, DiversiLab may be more appropriate for long-term epidemiological studies where less discrimination is needed.

Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping.

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.