Restless legs syndrome associated with exercise intolerance: Data from a retrospective observational clinical neuromuscular center study.

INTRODUCTION: Exercise intolerance (EI) is a frequent motive for seeking neuromuscular consultation and may be a sign of metabolic disease or, rarely, muscular dystrophy. The diagnosis is not established in many patients with a typical clinical presentation. Nevertheless, some of them complain of sleep disorders and more especially of restless legs syndrome (RLS). OBJECTIVE: The objective of our study was to estimate the frequency of RLS in patients presenting with EI. METHODS: Our retrospective observational study included all patients seen in the center from 2005 to 2011, who were subsequently investigated for EI in the neuromuscular department of the Caen University hospital. Data were collected on clinical RLS and muscular investigations (creatine kinase [CK], EMG, maximal exercise tests magnetic resonance imaging [MRI] and muscle biopsy obtained along with muscle exploration). RESULTS: Of the 318 patient records analyzed, 84 showed patients accurately complaining of EI. RLS was diagnosed in 25 of these patients (29.7%). This percentage was significantly higher (P<0.001) than found in the general population. Improvement was seen in 91.3% of the patients receiving specific treatment. CONCLUSION: RLS can sometimes present with pain, potentially worsening with exercise, inappropriately leading to a hypothesis of EI. Clinicians should thus explore the possible diagnosis of RLS when a muscular disease is not found in patients presenting with such symptoms.

Characterization of isolates of Listeria monocytogenes from sludge using pulsed-field gel electrophoresis and virulence assays.

AIMS: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. METHODS AND RESULTS: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque-forming assay and by mouse virulence assay. CONCLUSIONS: Our findings suggest that L. monocytogenes strains found in non-sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study dealing with the characterization of L. monocytogenes isolates from non-sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.

A Listeria monocytogenes strain is still virulent despite nonfunctional major virulence genes.

The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.

Prevalence of low-virulence Listeria monocytogenes strains from different foods and environments.

Various studies have demonstrated variations in the levels of virulence of different L. monocytogenes strains. In our laboratory, a plaque-forming assay followed by subcutaneous footpad inoculation of mice enabled us to estimate the prevalence of the low-virulence strains. This value fell from 16.3% to 1.7% with bacteria collected before 1994 and after 1997 respectively. This could be related to the modification in 1997 of the reference method EN ISO 11 290-1 of Listeria detection which recommended the use of polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) medium. The aim of this study was to determine whether the percentage of low-virulence strains detected has changed due to the modification of the detection method recommending the use of the ALOA medium. After analyzing 380 L. monocytogenes strains, no increase in the percentage of low-virulence strains could be detected. The prevalence reached only 2.6% (ten of the 380 strains tested). The low virulence of L. monocytogenes strains was not related to rare serotypes and was also observed in serotypes usually involved in human disease. Low-virulence strains were found in dairy, meat, ready-to-eat products and also in the environment, highlighting the absence of one specific source. These results are discussed in terms of detection methods and the definition of low virulence.

Prevalence and growth of Listeria monocytogenes in naturally contaminated cold-smoked salmon.

AIM: To investigate Listeria monocytogenes contamination and behaviour in naturally contaminated French cold-smoked salmon (CSS). METHOD AND RESULTS: Between 2001 and 2004, L. monocytogenes was detected in 104 of 1010 CSS packs, produced by nine French plants, with different prevalence (from 0% to 41%). The initial contamination, measured with a sensitive filtration method, was low (92% of contaminated products below 1 CFU g(-1)) and growth was limited. CONCLUSION: Growth was consistent with results of a predictive model including microbial competition. SIGNIFICANCE AND IMPACT OF THE STUDY: To be included in a quantitative risk assessment.

Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France.

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.

Outbreak of Salmonella enterica serotype Manhattan infection associated with meat products, France, 2005.

Between August 2005 and March 2006 in France, 69 cases of Salmonella enterica serotype Manhattan (Salmonella Manhattan) were reported, 51 (74%) of them from southeastern France. At the time of the alert (November 2005), 13 cases and 33 controls were interviewed. Cases were more likely than controls to have eaten pork sausages (OR=5.9, confidence interval CI [1.3; 26.9]) and beef (OR=9.3, CI [1.3; 68.6]). At the same time, 19 strains of Salmonella Manhattan isolated from meat products in southeastern France, reported to the French food safety agency (Afssa, Agence francaise de securite sanitaire des aliments) in September and November 2005, had an indistinguishable PFGE profile to the 7 human isolates of Salmonella Manhattan from the outbreak in southeastern France. Trace-back investigations revealed that pork samples came from one wholesaler whose pork products had tested positive for S. Manhattan during routine food testing in August 2005. This wholesaler supplied retail outlets in southeastern France. Additionally, a slaughterhouse supplying the wholesaler was inspected and widespread contamination with Salmonella spp. and S. Manhattan was found. Cooperation between the national agencies in charge of human health (Institut de veille sanitaire, InVS) and food safety (Afssa) allowed us to determine the most probable source of contamination and to take appropriate control measures.

Outbreak of Salmonella enterica serotype Manhattan infection associated with meat products, France, 2005.

Between August 2005 and March 2006 in France, 69 cases of Salmonella enterica serotype Manhattan (Salmonella Manhattan) were reported, 51 (74%) of them from southeastern France. At the time of the alert (November 2005), 13 cases and 33 controls were interviewed. Cases were more likely than controls to have eaten pork sausages (OR=5.9, confidence interval CI [1.3; 26.9]) and beef (OR=9.3, CI [1.3; 68.6]). At the same time, 19 strains of Salmonella Manhattan isolated from meat products in southeastern France, reported to the French food safety agency (Afssa, Agence française de sécurité sanitaire des aliments) in September and November 2005, had an indistinguishable PFGE profile to the 7 human isolates of Salmonella Manhattan from the outbreak in southeastern France. Trace-back investigations revealed that pork samples came from one wholesaler whose pork products had tested positive for S. Manhattan during routine food testing in August 2005. This wholesaler supplied retail outlets in southeastern France. Additionally, a slaughterhouse supplying the wholesaler was inspected and widespread contamination with Salmonella spp. and S. Manhattan was found. Cooperation between the national agencies in charge of human health (Institut de veille sanitaire, InVS) and food safety (Afssa) allowed us to determine the most probable source of contamination and to take appropriate control measures.

Investigation of specific substitutions in virulence genes characterizing phenotypic groups of low-virulence field strains of Listeria monocytogenes.

Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfADelta174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group.

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples.

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments.

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.

Comparison of five typing methods for the epidemiological study of Listeria monocytogenes.

Five typing methods were compared in a study designed to adapt a strategy for epidemiologically typing large numbers of Listeria monocytogenes strains. The methods studied were serotyping, electrophoretic typing of esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). Data were analysed by computer-assisted statistical analysis. Included in the analysis were 35 strains of L. monocytogenes, including 14 epidemic strains isolated during outbreaks in France in 1992 and 1993, and 21 strains isolated from food and the environment. Five serotypes, eight zymotypes, ten ribotypes, 13 RAPD patterns and 12 PFGE patterns were identified among the 35 strains. The most discriminating combination of typing methods was ribotyping and PFGE typing [27 types, discriminatory index (D.I.) = 0.978]. A factorial analysis of correspondence for each method differentiated the epidemic strains from the environmental strains. This study shows that computer-assisted statistical treatment of the data, combined with the use of discriminating typing methods, is a powerful tool for the epidemiological analysis of Listeria monocytogenes.

Molecular epidemiological tools for Salmonella Dublin typing.

A total of 32 strains of Salmonella Dublin recovered from cattle were differentiated by electrophoretic typing of their esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), arbitrarily primed PCR (AP-PCR) using five primers, PCR based on repetitive extragenic palindromic sequences (REP-PCR) and PCR based on enterobacterial repetitive intergenic consensus sequences (ERIC-PCR). ERIC-PCR and REP-PCR each gave one type, zymotyping gave three, AP-PCR gave five and ribotyping gave seven types. Combination of ribotyping and AP-PCR produced a total of 11 types, whereas 14 different types were obtained by all five methods. Thus a combination of several methods enhanced the discrimination of cattle-adapted strains among the genotypically homogeneous serovar Salmonella Dublin.