An amyotrophic lateral sclerosis hot spot in the French Alps associated with genotoxic fungi.

Between 1990 and 2018, 14 cases of amyotrophic lateral sclerosis (ALS) were diagnosed in residents of, and in visitors with second homes to, a mountainous hamlet in the French Alps. Systematic investigation revealed a socio-professional network that connected ALS cases. Genetic risk factors for ALS were excluded. Several known environmental factors were scrutinized and eliminated, notably lead and other chemical contaminants in soil, water or home-grown vegetation used for food, radon and electromagnetic fields. Some lifestyle-related behavioral risk factors were identified: Prior to clinical onset of motor neuron disease, some patients had a high degree of athleticism and smoked tobacco. Recent investigations on site, based on a new hypothesis, showed that all patients had ingested wild mushrooms, notably poisonous False Morels. Half of the ALS cohort reported acute illness following Gyromitra gigas mushroom consumption. This finding supports the hypothesis that genotoxins of fungal origin may induce motor neuron degeneration.

Safety assessment of Gram-negative bacteria associated with traditional French cheeses.

Twenty Gram-negative bacterial (GNB) strains were selected based on the biodiversity previously observed in French traditional cheeses and their safety was assessed considering various safety criteria. For the majority of tested GNB strains, only gastric stress at pH 2 (vs pH 4) resulted in low survival and no regrowth after an additional simulated gastro-intestinal stress. Presence of milk was shown to be rarely protective. The majority of strains was resistant to human serum and had a low level of adherence to Caco-2 cells. When tested for virulence in Galleria mellonella larvae, GNB strains had LD 50 values similar to that of safe controls. However, four strains, Hafnia paralvei 920, Proteus sp. (close to P. hauseri) UCMA 3780, Providencia heimbachae GR4, and Morganella morganii 3A2A were highly toxic to the larvae, which suggests the presence of potential virulent factors in these strains. Noteworthy, to our knowledge, no foodborne intoxication or outbreak has been reported so far for any of the GNB belonging to the genera/species associated with the tested strains. The role of multiple dynamic interactions between cheese microbiota and GIT barriers could be key factors explaining safe consumption of the corresponding cheeses.

Usefulness of FTIR spectroscopy to distinguish rough and smooth variants of Lactobacillus farciminis CNCM-I-3699.

In this study, the potential of Fourier transform infrared (FTIR) spectroscopy for assessing putative biochemical and structural differences between the two variants, rough (R) and smooth (S), of Lactobacillus farciminis CNCM-I-3699, a pleomorphic strain, was investigated. The main differences observed were localized in the polysaccharide (1200-900 cm-1) and protein (1700-1500 cm-1) regions. Based on spectral information in these two spectral ranges, clustering resulted in a dendrogram that showed a clear discrimination between both morphotypes. Significant increases in favor of morphotype S compared to R at specific wavenumbers for polysaccharides (22.18% vs. 5.24% at 1068 cm-1) and capsular polysaccharides (16% vs. 13.17% at 1048 cm-1) were recorded. Compared to S, the morphotype R exhibits a 1.27-fold higher signal at the wavenumber of 1637 cm-1 assigned to the amide I ß-sheet and a 2.71-fold higher signal at the wavenumber of 1513 cm-1 assigned to the tyrosine involved in the ß-sheet arrangement of proteins. The FTIR analysis is efficient to separate and give data on mainly surface component differences observed previously between S colony morphotype (ropy and exopolysaccharide positive) and the R colony morphotype (non-ropy but highly autoaggregative).

Draft Genome Sequence of Enterococcus mundtii QAUEM2808, Isolated from Dahi, a Fermented Milk Product.

Enterococcus mundtii QAUEM2808 has been isolated from dahi, an indigenous fermented milk product of Pakistan. Here, we report the draft genome sequence for this strain, which consists of 160 contigs corresponding to 2,957,514 bp and a G+C content of 38.5%.

Genome Sequence of Rough and Smooth Variants of Pleomorphic Strain Lactobacillus farciminis CNCM-I-3699.

The probiotic Lactobacillus farciminis CNCM-I-3699 is a pleomorphic strain exhibiting smooth and rough variants. We report their complete genomes consisting of a chromosome of 2, 4 Mb and a plasmid of 6,417 bp. The smooth variant differs by the presence of an additional plasmid of 35,418 bp.

Genome Sequence of Lactobacillus rhamnosus Strain CNCM I-3698.

Lactobacillus rhamnosus CNCM I-3698 is a commercially available probiotic that is used in animal feed as an additive. Here, we announce the draft genome sequence for this strain, consisting of 71 contigs corresponding to 2,966,480 bp and a G+C content of 46.69%.

Overview of differences between microbial feed additives and probiotics for food regarding regulation, growth promotion effects and health properties and consequences for extrapolation of farm animal results to humans.

For many years, microbial adjuncts have been used to supplement the diets of farm animals and humans. They have evolved since the 1990s to become known as probiotics, i.e. functional food with health benefits. After the discovery of a possible link between manipulation of gut microflora in mice and obesity, a focus on the use of these beneficial microbes that act on gut microflora in animal farming was undertaken and compared with the use of probiotics for food. Beneficial microbes added to feed are classified at a regulatory level as zootechnical additives, in the category of gut flora stabilizers for healthy animals and are regulated up to strain level in Europe. Intended effects are improvement of performance characteristics, which are strain dependent and growth enhancement is not a prerequisite. In fact, increase of body weight is not commonly reported and its frequency is around 25% of the published data examined here. However, when a Body Weight Gain (BWG) was found in the literature, it was generally moderate (lower than or close to 10%) and this over a reduced period of their short industrial life. When it was higher than 10%, it could be explained as an indirect consequence of the alleviation of the weight losses linked to stressful intensive rearing conditions or health deficiency. However, regulations on feed do not consider the health effects because animals are supposed to be healthy, so there is no requirement for reporting healthy effects in the standard European dossier. The regulations governing the addition of beneficial microorganisms to food are less stringent than for feed and no dossier is required if a species has a Qualified Presumption of Safety status. The microbial strain marketed is not submitted to any regulation and its properties (including BWG) do not need to be studied. Only claims for functional or healthy properties are regulated and again growth effect is not included. However, recent studies on probiotic effects showed that BWG could also be observed in humans, or not, according to species and strains. Determining the significance of farm animal results for extrapolation to humans, especially regarding body weight improvement, was not easy because they do not use the same microbial strains nor always the same species. Furthermore, the framework for the management of microbials added to feed or to food differ, especially with regard to goal, timescale and lifestyle. So no one can exclude the possibility that beneficial microorganisms having probiotic effects may have long-term effects in humans that cannot be seen to date in animals, where short-term use is the rule. A possible link to obesity cannot be excluded in relation to timescale, species and strain specificity. To conclude, beneficial microorganisms added in feed are key factors stringently regulated for short-term improvement of zootechnical performances in animals and their use does not entirely parallel that of human probiotics. So extrapolation of farm animal results to humans is biased and not sufficient to be conclusive regarding the existence or not of a link between probiotics and obesity. From a toxicological and nutritional point of view and considering recent findings on a link between antibiotic use in early life and excessive risk of becoming overweight, one suggestion is to study the at-risk population in Europe, pregnant women and their babies before and after birth and during early childhood, in an epidemiological long-term cohort survey.

From undefined red smear cheese consortia to minimal model communities both exhibiting similar anti-listerial activity on a cheese-like matrix.

Starting from one undefined cheese smear consortium exhibiting anti-listerial activity (signal) at 15 °C, 50 yeasts and 39 bacteria were identified by partial rDNA sequencing. Construction of microbial communities was done either by addition or by erosion approach with the aim to obtain minimal communities having similar signal to that of the initial smear. The signal of these microbial communities was monitored in cheese microcosm for 14 days under ripening conditions. In the addition scheme, strains having significant signals were mixed step by step. Five-member communities, obtained by addition of a Gram negative bacterium to two yeasts and two Gram positive bacteria, enhanced the signal dramatically contrary to six-member communities including two Gram negative bacteria. In the erosion approach, a progressive reduction of 89 initial strains was performed. While intermediate communities (89, 44 and 22 members) exhibited a lower signal than initial smear consortium, eleven- and six-member communities gave a signal almost as efficient. It was noteworthy that the final minimal model communities obtained by erosion and addition approaches both had anti-listerial activity while consisting of different strains. In conclusion, some minimal model communities can have higher anti-listerial effectiveness than individual strains or the initial 89 micro-organisms from smear. Thus, microbial interactions are involved in the production and modulation of anti-listerial signals in cheese surface communities.

[Evaluation of Antilles fish ciguatoxicity by mouse and chick bioassays]. / Evaluation de la ciguatoxicité de poissons des antilles par les bioessais souris et poussin.

Ciguatera is a common seafood poisoning in Western Atlantic and French West Indies. Ciguatera fish poisoning in the Caribbean is a public health problem. A toxicological study was carried out on 178 Caribbean fish specimens (26 species) captured off Guadeloupe and Saint Barthelemy between 1993 and 1999. The mouse bioassay and the chick feeding test were used to control fish edibility. Ciguatoxins presence was assumed when symptomatology was typical of ciguatera in mouse and chick. Fishes were classified in three groups: non toxic fish (edible), low toxic fish (not edible) and toxic fish (not edible). 75% of fishes were non toxic. Toxic fish specimens belonged to four families of high trophic level carnivores: Carangidae, Lutjanidae, Serranidae et Sphyraenidae. Percentages of toxic fishes to humans reached 55% for Caranx latus and 33% for Caranx bartholomaei and Caranx lugubris. Only a significant correlation between weight and toxicity was only found for C. latus and snappers. Small carnivorous groupers (Serranidae) were also toxic. Atoxic fish species were (a) pelagic fish (Coryphaena hippurus, Auxis thazard and Euthynnus pelamis), (b) invertebrates feeders (Malacanthus plumieri, Balistes vetula), (c) small high-risk fish or (d) fish of edible benthic fish families. Liver of four fishes (Mycteroperca venenosa, Caranx bartholomaei, Seriola rivoliana, Gymnothorax funebris) contained ciguatoxins at a significant level although their flesh was safe. This study confirms the usefulness of mouse and chick bioassays for sanitary control of fish.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies.

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a black jack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, > or = 1 and > 1 MUg g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H]+ ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

Ciguatera fish poisoning in the Caribbean islands and Western Atlantic.

Ciguatera fish poisoning (ciguatera), a common poisoning caused by fish ingestion, is reviewed in the Western Atlantic and the Caribbean waters. It is endemic from Florida coasts (northern limit) to Martinique Island (southern limit), with outbreaks occurring from time to time. In the Caribbean, ciguatera causes a polymorphic syndrome with gastrointestinal, cardiovascular, and neurological signs and symptoms. Neurological and muscular dysfunctions can be treated by intravenous injection of D-mannitol. The lipid-soluble toxins involved are ciguatoxins that are likely produced by the dinoflagellate Gambierdiscus toxicus. G. toxicus strains are endemic in the Caribbean Sea and in theWestern Atlantic. Although it is likely that blooms of G. toxicus are ingested by herbivorous fishes, they are not implicated in ciguatera in the Caribbean. Rather, large carnivores (barracudas, jacks, snappers, groupers), consumers of smaller benthic fish, are often involved in ciguatera. Fish toxicity depends on fishing area and depth, fish size and tissues, and climatic disturbances. Ciguatoxins have been isolated and purified from Caribbean fish species. The structure of two epimers, C-CTX-1 and C-CTX-2 from horse-eye jack, comprise 14 trans-fused ether-linked rings and a hemiketal in terminal ring. Caribbean ciguatoxins are mainly detected in the laboratory by chicken, mouse, mosquito, or cell bioassays, and by analytical HPLC/tandem mass spectrometry down to parts per billion (ppb). A ciguatera management plan that integrates epidemiology, treatment, and a simple method of detection is required to ensure the protection of consumers.

Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed.

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.

HPLC/tandem electrospray mass spectrometry for the determination of Sub-ppb levels of Pacific and Caribbean ciguatoxins in crude extracts of fish.

Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels > 0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of > 0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh.

Isolation and characterisation of Caribbean ciguatoxins from the horse-eye jack (Caranx latus).

The toxins involved in ciguatera (fish poisoning) in the Caribbean Sea were isolated from Caranx latus, a pelagic fish often implicated in ciguatera in the Caribbean region, and purified by mouse bioassay directed fractionation. Five toxins were separated by reverse-phase high-performance liquid chromatography (HPLC). In order of increasing hydrophobicity, these toxins included a sleep-inducing fraction (< 1% of total toxicity), a major Caribbean ciguatoxin (C-CTX-1, 65% of toxicity), a minor Caribbean ciguatoxin (C-CTX-2, 13% of toxicity), a minor toxin (approximately 1% of toxicity) and a hydrophobic, fast-acting toxin (approximately 19% of toxicity). The i.p. injection into mice of each toxin induced signs typical of site-5 sodium channel activator toxins such as the Pacific ciguatoxins and brevetoxins. C-CTX-1 and C-CTX-2 were purified to homogeneity (LD50 = 3.6 and approximately 1 microgram/kg, respectively) and subjected to ion spray mass spectrometry. Both lost up to five H2O molecules and each had a [M+H]+ ion, m/z 1141.7, suggesting that C-CTX-1 and -2 are diastereomers that differ from the Pacific family of ciguatoxins. Turbo-assisted HPLC-mass spectrometry identified C-CTX-1, C-CTX-2 and three C-CTX-1-related compounds in an enriched fraction but no Pacific ciguatoxins were detected. The presence of different families of ciguatoxins in ciguateric fish from the Caribbean Sea and Pacific Ocean probably underlies the clinical differences in the ciguatera syndrome reported in these two regions. A Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus, is suspected as source of these ciguatoxins. The extent to which these toxins are biotransformed as they pass through the marine food chain remains to be determined.

Effect of an unknown toxin isolated from Dinophysis sp.-contaminated French mussels, on the electrical and mechanical activity of frog heart.

The effects of an unknown toxin, isolated along with okadaic acid from the hepatopancreas of French mussels contaminated by Dinophysis sp., producing ataxia, neurologic symptoms, bradycardia, arrhythmias, electrocardiographic changes, and cardiac arrest, have been studied in terms of the electrical and mechanical activity of frog atrial heart muscle. The toxin, in a dose-dependent manner, decreased the amplitude of the stimulated peak tension of isolated fibers. The toxin (1-36 micrograms/ml) did not modify the membrane resting potential but decreased the amplitude of the plateau and shortened the duration of the action potential. The toxin inhibited the Cd-sensitive L-type Ca current and increased a 4-aminopyridine-sensitive outward current in voltage-clamped cardiac myocytes. The data show that the cardiac effect of the toxin is markedly different from that of okadaic acid.

Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate.

A colorimetric phosphatase-inhibition bioassay was developed for the quantitative measurement of okadaic acid (OA) the main diarrhetic toxin responsible for diarrhetic shellfish poisoning. The assay used an artificial substrate, paranitrophenylphosphate, and a semi-purified protein phosphatase PP2Ac containing extract prepared from rabbit muscle. Calibration dose-inhibition curves were constructed using standard OA and they permitted easy determination of the enzyme concentration Et in their linear portion. In the range of linearity, the slope increased when Et decreased, thus giving a detecting limit of 0.04 pmol in the reaction mixture (1 ml). The lowest assayable concentration of OA was 4 ng/ml in aqueous solutions and 40 ng/ml (i.e., 100 ng of OA per g of mussel tissue) in crude methanol mussels extracts. The intra and interassay coefficients of variation in the measurement of OA for the toxin spiked aqueous samples averaged, respectively, 7.7% and 3.7%, and interexperiments coefficients of variation for the toxin spiked mussel extracts averaged 4.6%. The presence of OA was ascertained by a method in which one assay was performed at two or three different levels of enzyme concentration. The rapidity, accuracy, reproducibility, specificity, and simplicity of the procedure provides a simple way to assay okadaic acid in buffered or complex solutions.

Studies on the detection of okadaic acid in mussels: preliminary comparison of bioassays.

Diarrheic toxins, especially okadaic acid, are detected nearly every year in mussels on French coasts. The monitoring network determines the toxicity of these shellfish by using a mouse test now considered unsatisfactory from an ethical point of view. Two alternative methods have been investigated: the daphnia test, for which there is a standardized method used routinely in ecotoxicology, and a cytotoxicity test on the KB cell line developed for this study. Using the same samples, the results of these two tests were compared with those obtained by chemical analysis (HPLC okadaic acid assay) or the mouse test. Linear regression studies showed that results for the two bioassays were well correlated with those for HPLC or the mouse test.

Cooked mussels contaminated by Dinophysis sp.: a source of okadaic acid.

A method was developed for fractionation and isolation of toxic components present in extracts prepared from Dinophysis-contaminated mussels. The major toxin present in French mussels was identified as okadaic acid by its chromatographic properties and spectral data. Large amounts of mussel tissue (digestive glands and remaining meat) can be treated easily if they are cooked, or cooked and dried and are useful for isolating significant amounts of okadaic acid.

The use of Daphnia magna for detection of okadaic acid in mussel extracts.

Okadaic acid (OA), the main toxin responsible for diarrheic shellfish poisoning (DSP) has toxicity for Daphnia magna (EC50 = 15 +/- 1.8 micrograms/l). A Daphnia bioassay was developed and used to analyse okadaic acid in mussel extracts. A linear correlation was found between OA concentration determined by the Daphnia bioassay and by HPLC assay (r = 0.85; p < 0.001). The Daphnia bioassay can measure OA levels 10 times below the threshold of the mouse bioassay method. It is an inexpensive sensitive tool which can be used in replacement of mouse bioassay for the screening of OA and some co-extracting toxins in mussel extracts.

Fractionation and purification of some muscular and visceral ciguatoxins extracted from Carribean fish.

1. Cigautoxins (CTX) were extracted from flesh and viscera of seven large roving predatory fishes: Caranx bartholomaei, Caranx latus, Seriola dumerili, Alectis crinitus, Scomberomorus cavalla, Sphyraena barracuda and Gymnothorax funebris. 2. Generally each extract consisted of close-related CTX which were separated according to their polarity by Florisil column chromatography into a fast-acting CTX containing group and a slow-acting CTX containing group. 3. The shortest survival time of mice (ts) was low for the former group (less than 10 min) and high for the latter (greater than or equal to 29 min). 4. The level of purity had no influence on the range of ts values. The presence of these two CTX groups in different extracts did not from experimental conditions. 5. Attempts to convert fast-acting CTX to slow-acting CTX and vice-versa were negative. G. funebris and S. barracuda had an especially high content of unstable fast-acting CTX. 6. Purification of the slow-acting CTX was achieved by fast elution chromatography and Sephadex LH20 gel filtration. 7. The ts values of these CTX were identical for five species (40-44 min) but not for S. barracuda (29-32 min). 8. Thus ciguatoxic extracts from Caribbean fish were composed of several close-related CTX.