Sensitive enumeration of Listeria monocytogenes and other Listeria species in various naturally contaminated matrices using a membrane filtration method.

For the enumeration of Listeria monocytogenes (L. monocytogenes) in food, a sensitive enumeration method has been recently developed. This method is based on a membrane filtration of the food suspension followed by transfer of the filter on a selective medium to enumerate L. monocytogenes. An evaluation of this method was performed with several categories of foods naturally contaminated with L. monocytogenes. The results obtained with this technique were compared with those obtained from the modified reference EN ISO 11290-2 method for the enumeration of L. monocytogenes in food, and are found to provide more precise results. In most cases, the filtration method enabled to examine a greater quantity of food thus greatly improving the sensitivity of the enumeration. However, it was hardly applicable to some food categories because of filtration problems and background microbiota interference.

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.

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, 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.

Anatomic study of middle fossa approach landmarks using an image guidance system.

OBJECTIVES: We sought to verify the relevance and reliability of the main temporal bone anatomic landmarks commonly used for the middle fossa approach and to test the use of digital technology to perform an accurate analysis of these landmarks. METHODS: Ten fresh cadaveric temporal bones (5 heads) were analyzed by both computed tomographic imaging and dissection procedures, with the help of an image guidance system (DigiPointeur). Eight landmarks, which were selected for their wide citation in the otoneurosurgical literature, were studied. RESULTS: Of the 8 landmarks studied, we obtained results similar to previously published data for 2 (the distance between the temporal squama and the internal auditory meatus [IAM] fundus, and the distance between the top of the anterior semicircular canal [ASCC] arch and the IAM fundus), but divergent measurements for the other 6 (the distance between the temporal squama and the arcuate eminence; the distance between the top of the ASCC arch and the cochlear promontory; the IAM roof thickness; the angle formed by the ASCC arch plane orientation and the IAM axis; the angle formed by the head of the malleus, the geniculate ganglion, and the IAM fundus; and the correspondence between the arcuate eminence and the top of the ASCC arch). CONCLUSIONS: The use of an image guidance system allowed us to make sharp and precise measurements of the main anatomic landmarks used during a middle fossa approach. We found 6 measurements to be different from those reported in previously published data out of the 8 landmarks studied.

Validation of standard method EN ISO 19343 for the detection and quantification of histamine in fish and fishery products using high-performance liquid chromatography.

In contaminated fish, bacterial decarboxylases produce histamine from histidine, thereby causing scombroid fish poisoning. European Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs requires using a fully validated, standardized reference HPLC method for detecting and quantifying histamine. After optimizing this reference method for the quantification of histamine in fish muscle, we organized an inter-laboratory study in 2013 across nine laboratories from seven European countries using defined criteria of method performance. The optimized, validated method was standardized (Standard EN ISO19343) as part of Mandate M381 from the European Commission to the European Committee for Standardization (CEN), signed in December 2010. The standard method was validated for three types of foodstuffs (fish with enzymatic maturation, fish without enzymatic maturation and fish sauce).

Validation of standard method EN ISO 11290 - Part 1 - Detection of Listeria monocytogenes in food.

The reference method for the detection and enumeration of L. monocytogenes in food (Standards EN ISO 11290-1&2) has been validated by inter-laboratory studies in the frame of the Mandate M381 from European Commission to CEN. In this paper, the collaborative studies led in 2013 on 5 matrices (cold-smoked salmon, milk powdered infant food formula, vegetables, environment, and cheese) to validate the recently revised Standard EN ISO 11290-Part 1 are reported. According to the results obtained, the revised Standard EN ISO 11290-1 can be considered as a good method for the detection of L. monocytogenes in foods and food processing environments, in particular for the matrices included in the study. According to the matrices, the sensitivity rate varied from 91.1% to 100%, and the specificity rate varied from 97.6% to 100%. Positive samples were most often detected after 24 h half-Fraser enrichment.

Validation of standard method EN ISO 11290 - Part 2 for the enumeration of Listeria monocytogenes in food.

The reference method for the detection and enumeration of L. monocytogenes in food (Standards EN ISO 11290-1&2) have been validated by inter-laboratory studies in the frame of the Mandate M381 from European Commission to CEN. In this paper, the inter-laboratory studies led in 2013 on 5 matrices (cold-smoked salmon, milk powdered infant food formula, vegetables, environment, and cheese) to validate Standard EN ISO 11290-2 are reported. According to the results obtained, the method of the revised Standard EN ISO 11290-2 can be considered as a good method for the enumeration of L. monocytogenes in foods and food processing environment, in particular for the matrices included in the study. Values of repeatability and reproducibility standard deviations can be considered satisfactory for this type of method with a confirmation stage, since most of them were below 0.3 log10, also at low levels, close to the regulatory limit of 100 CFU/g.

Evaluation of an enumeration method for Listeria monocytogenes at low contamination levels in cold-smoked salmon.

For the enumeration of Listeria monocytogenes in cold-smoked salmon, a sensitive enumeration method, based on membrane filtration followed by transfer of the filter on a selective medium has been recently developed (Gnanou Besse et al., 2004, A contribution to the improvement of L. monocytogenes enumeration in cold-smoked salmon. International Journal of Food Microbiology, 91, 119-127). The aim of the study was to assess the performance of this enumeration method through an inter-laboratory study, using cold-smoked salmon artificially contaminated at 2 different levels (approximately 0.6 and 1.6 log10 CFU g(-1)). A reproducibility standard deviation of 0.23 log10 CFU g(-1)and 0.15 log10 CFU g(-1) was obtained for the method respectively at the lower level and the higher level. Under certain conditions, the uncertainty of measurement can be derived from the method reproducibility standard deviation and was calculated to be 0.46 log10 CFU g(-1) for the lower contamination level and 0.30 log10 CFU g(-1) for the higher contamination level. These values can be considered as satisfactory for such low contamination levels.

Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories.

Enterobacter sakazakii is an occasional contaminant of powdered infant formula that can cause rare but severe foodborne infections in infants. To determine optimal methods for the detection and identification of E. sakazakii, 38 naturally contaminated samples from infant formulae factories were analyzed by two PCR-based methods and by a method (TS 22964/RM 210) developed by the International Organization for Standardization and the International Dairy Federation (ISO-IDF) using three different commercial chromogenic agars. The ISO-IDF method includes two enrichment steps, plating of the second enrichment broth on E. sakazakii isolation agar (a chromogenic selective agar), picking of five typical colonies for transfer onto tryptone soy agar, and subsequent confirmation of yellow-pigmented colonies by biochemical characterization. Twenty-two of the 38 samples were positive by the culture method. E. sakazakii isolation agar (ESIA; AES Laboratoires), COMPASS agar (Biokar Diagnostics), and Druggan-Forsythe-Iversen agar (Oxoid) compared favorably with violet red bile glucose agar (VRBG, a selective medium for Enterobacteriaceae), with positive predictive values of 86.96, 88, and 74.07%, respectively, in contrast to 47.83% for VRBG. One additional positive sample was detected using the nonpatented real-time PCR method evaluated, and those results were in 97.3% concordance with the ISO-IDF results. Some discrepancies between the results of the DuPont Qualicon BAX system and those of the ISO-IDF method could be explained by heterogeneity of contamination and sampling. Thus, both PCR-based systems were suitable for detecting and specifically identifying E. sakazakii within 1 to 2 days, and COMPASS agar and ESIA could be used interchangeably as a first-step medium to isolate presumptive E. sakazakii colonies.

Interlaboratory validation of the Vidas SET2 kit for detection of staphylococcal enterotoxins in milk products.

An earlier intralaboratory validation study based on the EN ISO 16140 Standard conducted by the Community Reference Laboratory for coagulase-positive staphylococci including Staphylocococcus aureus showed that, after an extraction step using dialysis concentration, the Vidas SET2 detection kit could be used to screen staphylococcal enterotoxins in milk and milk products. In order to fully validate Vidas SET2, an interlaboratory study was organized. Six freeze-dried samples and 3 ready-to-use concentrated extracts were analyzed by 21 laboratories according to the method, including a detection with Vidas SET2. Results did not show false-positive or -negative results. Accordance and concordance parameters were equal to 100%, corresponding to a concordance odds ratio of 1. This interlaboratory study confirmed the satisfactory outcome of the preliminary tests and of the intralaboratory study performed previously. The Vidas SET2 detection kit can be used as a method for the detection of staphylococcal enterotoxins in milk and milk products as well as the Transia Plate SET detection kit in the European screening method for official control purposes, after an extraction step followed by dialysis concentration.

Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables.

Salad vegetables exposed to fecal contamination may cause outbreaks of hepatitis or gastro-enteritis if they are eaten raw. A procedure, based on elution with phosphate-buffered saline and concentration by filtration through membrane filters, was developed for the recovery of enteric viruses from salad leaves. The method was evaluated using lettuce leaves inoculated with hepatitis A virus (HAV), poliovirus, and MS2 bacteriophage. In addition, this method was validated by an intra-laboratory study using leaves of various salad vegetables inoculated with MS2 phage. The French standard NF V 03-110 was used to establish the general principle and the technical protocol of the validation procedure. Linear regression models describing the quantitative reactions were good fits to data in the whole range of viral concentrations tested, which was from about 1 to 4 log plaque-forming units (PFU) per 25 g of lettuce. The fractions of inoculated viruses recovered were estimated to be about 64% for HAV, 18% for poliovirus, and 29% for MS2. No significant effect of the food matrix was found using various types of salad vegetable (butter lettuce, iceberg lettuce, romaine lettuce, witloof chicory, curly endive, corn salad, rocket and watercress). Moreover, the variance of the results was constant for all levels of virus contamination within the experimental range. Intermediate reproducibility experiments were also performed to allow calculation of the uncertainty factor, which was found to be 0.58 log PFU/25 g. When used in association with phage enumeration, this validated procedure is rapid enough to be used for screening salad vegetables for evaluation of the efficacy of processes for control of pathogenic microorganisms on such foods.

Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

Specific activation of operculum 3 (OP3) brain region during provoked tinnitus-related phantom auditory perceptions in humans.

The phantom sound perception mechanism by which a sound perception occurs without any external sound source is still enigmatic. According to our previous fMRI study, a small region in the parietal operculum 3 was hyperactivated as a function of tinnitus periodicity in subjects with acoustic trauma tinnitus sequelae. This region was localized in the vicinity of neural correlates of middle-ear tympano-ossicular chain movements due to pressure variations. Disturbed proprioceptors are known to trigger illusory perceptions; therefore, we hypothesized that a disturbance of middle-ear proprioceptors may originate phantom sound perceptions. We designed an fMRI study that aimed to stimulate middle-ear proprioceptors by repetitive vibrations using various rates of click trains. In this study, we report that exposure to specific rates of stimuli for a few minutes at comfortable intensity level in healthy subjects distinctly triggered transient tinnitus-like aftereffects. The fMRI neural correlates of the aftereffects were unequivocally localized in the same parietal region as in acoustic trauma tinnitus sufferers. Our results strongly suggest that a middle-ear kinesthetic/proprioceptive illusion exists at the origin of acoustic trauma tinnitus via a somatosensory pathway encompassing the trigeminal system.

Experimental evaluation of different precision criteria applicable to microbiological counting methods.

The dispersion of microbiological counting measurements, when repeating the analysis on the same material both within a laboratory (repeatability) and between laboratories (reproducibility) can be characterized by the organization of interlaboratory studies, where several sets of identical test materials are sent to several laboratories. Using the example of data generated by an interlaboratory study on enumeration of Listeria monocytogenes in foods by the standardized reference method (colony-count technique), 2 types of robust estimators of reproducibility standard deviations, based on the median, were examined, in comparison with the classical estimators, based on the mean. Experimental evaluation indicated that the 3 approaches gave consistent results for most of the combinations. The usual log10 transformation of the enumeration results was also questioned before these calculations were conducted.

Applicability of the draft standard method for the detection of Escherichia coli O157 in dairy products.

According to the draft standard method, EN ISO 16654 for the detection of Escherichia coli O157 in foods, samples of milk products inoculated with E. coli O157 were cultured in modified Tryptone Soya Broth supplemented with novobiocin. After immuno-magnetic separation (IMS) of the micro-organisms with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti E. coli O157, Dynal), the enrichment broth was subcultured onto both Cefixim Tellurite Sorbitol MacConkey agar and CHROMagar O157. IMS alone appeared not to be very specific to E. coli O157; however, IMS combined with CT-SMAC plating enabled a greater exclusivity. The method displayed a low limit of detection (1-2 cfu/25 g) of E. coli O157 strains in milk products after only 6 h of incubation of the enrichment broth. However, the detection was affected by storage of the inoculum at 4 degrees C, and another 12 to 18 h of incubation was necessary to recover potentially stressed cells.

A contribution to the improvement of Listeria monocytogenes enumeration in cold-smoked salmon.

For the enumeration of Listeria monocytogenes in food, a sensitive enumeration method based on membrane filtration followed by transfer of the filter to a selective medium has been developed. This study was carried out with cold-smoked salmon, a product likely to be contaminated with L. monocytogenes. The operating protocol utilizes three filtration runs in parallel (5, 15 and 30 ml) of a 1 in 10 dilution of the salmon suspension through 0.45-microm pore-size cellulose ester membranes, and then culture of the filters on Aloa agar (AES Laboratoires, Combourg, France). The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L. monocytogenes from both artificially and naturally contaminated cold-smoked salmon. Moreover, for several samples contaminated at low levels, L. monocytogenes could be recovered only by the filtration method. The examination of increasing volumes of salmon suspension enabled readable results to be obtained for all levels of L. monocytogenes and competitive microflora investigated. In most cases, the optimised operating protocol enabled 5.1 g of salmon to be examined, instead of 0.01-0.1 g with the reference EN ISO 11290-2 method, thus improving the sensitivity of the method.

First proficiency testing to evaluate the ability of European Union National Reference Laboratories to detect staphylococcal enterotoxins in milk products.

The European Commission has designed a network of European Union-National Reference Laboratories (EU-NRLs), coordinated by a Community Reference Laboratory (CRL), for control of hygiene of milk and milk products (Council Directive 92/46/ECC). As a common contaminant of milk and milk products such as cheese, staphylococcal enterotoxins are often involved in human outbreaks and should be monitored regularly. The main tasks of the EU-CRLs were to select and transfer to the EU-NRLs a reference method for detection of enterotoxins, and to set up proficiency testing to evaluate the competency of the European laboratory network. The first interlaboratory exercise was performed on samples of freeze-dried cheese inoculated with 2 levels of staphylococcal enterotoxins (0.1 and 0.25 ng/g) and on an uninoculated control. These levels were chosen considering the EU regulation for staphylococcal enterotoxins in milk and milk products and the limit of detection of the enzyme-linked immunosorbent assay test recommended in the reference method. The trial was conducted according to the recommendations of ISO Guide 43. Results produced by laboratories were compiled and compared through statistical analysis. Except for data from 2 laboratories for the uninoculated control and cheese inoculated at 0.1 ng/g, all laboratories produced satisfactory results, showing the ability of the EU-NRL network to monitor the enterotoxin contaminant.

Building a molecular Listeria monocytogenes database to centralize and share PFGE typing data from food, environmental and animal strains throughout Europe.

The European Union Reference Laboratory (EURL) for Listeria monocytogenes (Lm) collaborates with a network of 35 National Reference Laboratories (NRLs) throughout Europe. Most of these NRLs are in charge of detecting and typing Lm strains from food, environment and animals, which are isolated nationally. The past few years EURL activities have enabled NRLs to reinforce typing capabilities according to standardised protocols. Consequently the need to exchange typing data within the NRL network has emerged. That is why the EURL has recently set up a EURL Lm Database (EURL Lm DB). Each NRL contributes data, which is then shared within the network. Data include strain-typing-results (PFGE and serotyping) and epidemiological information on the strains. This article describes (1) the EURL typing activities that led to the creation of the EURL Lm DB, (2) the different steps involved in developing the EURL Lm DB, and (3) the usefulness of this database for public health. The combined use of this database, with databases on human strains, is being integrated into the European surveillance system of Lm strains circulating throughout Europe. It should improve the detection of this pathogen and provide support for outbreak investigations.