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.

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.

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.

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%.

[Fractionation of ciguatoxic lipid extracts in alkaline medium]. / Fractionnement d'extraits lipidiques ciguatoxiques en milieu alcalin

A modification of the extraction procedure for obtaining ciguatoxic substances from the tissues of various poisonous reef fish has been developed. Rapid alkali treatment of the residue obtained at an early stage is efficient in separating the ciguatoxic fraction from toxic impurities which interfere with pharmacological tests. It is thus possibles to carry out a more precise dosage of the material separated in this way. Alkali treatment does not affect either the physiological activity in cats and mice of the ciguatoxic substances nor their behaviour in a preparative thin layer chromatography in a benzene-butanol system.

[Heterogeneity of ciguatoxins extracted from fish caught in the French Antilles]. / Hétérogénéité des ciguatoxines extraites de poissons péchés aux Antilles françaises.

Ciguatoxin-like substances were extracted from the viscera or the flesh of eight Caribbean fish species, including small invertebrate feeders and large carnivores. The had similar properties, i.e. pharmacological action, solubility, chromatographic behaviour on silicic acid or Sephadex LH 20 column, stability in a weak acid solution and instability in alkaline medium. However, Florisil column and thin-layer chromatography showed different ciguatoxins whose number depended on tissue or species but not on fish trophic level. Less polar ciguatoxins appeared in salted and dried flesh. Thus, fish ciguatoxins are believed to be closely related substances, possibly changing in structure according to particular experimental conditions.

[Demonstration of a liposoluble ciguateric toxin in Caranx bartholomaei caught in the French West Indies]. / Mise en évidence d'une toxine liposoluble de type ciguatérique chez Caranx bartholomaei pêché aux Antilles françaises.

This report deals with some of the biological and chemical properties of a liposoluble poison extracted from Caranx bartholomaei (yellow jack), a toxic fish from the French West Indies (St Barth's island). Within a single fish, poison concentration is higher in the viscera; toxicity level is uniform throughout the flesh. In the few specimens tested, liver toxicity variations parallel those of flesh. The poison is heat stable so that cooking does not impair the toxicity. It is soluble in acetone, diethyl ether, chloroform, benzene, methanol and ethanol, but insoluble in n. hexane. Crude toxin injection or ingestion induces ciguateric disease in cats, mice or newly born chicken. Crude toxin stability is good after 30 minutes at 90 degrees C in a 0,5 N solution of a weak acid but not of a weak base. In the same conditions, loss of activity is nearly complete with a strong acid or a strong base. Moreover, rapid alkali treatment at room temperature destroys more than 50 per cent of the toxicity. The toxin is eluted by chloroform-methanol (9:1) from a silicic acid column and by acetone-methanol (9,5:0,5) from a Florisil column. DEAE cellulose column chromatography clearly separates the toxin into two lethal components; but thin layer chromatography of crude or fractionated toxin indicates only one toxic band in three different solvent systems. As a conclusion, in spite of a few differences related to instability in alkalin medium and elution from DEAE cellulose, this poison is quite similar to those carried by ciguatoxic fishes from the Pacific area. The differences noted above could be accounted for either by a specific metabolism of the species studied or by differences in the causative agent due to geographical location, or both.

Chick feeding test: a simple system to detect ciguatoxin.

Chick poisoning induced by oral administration of toxic fish tissues or extracts gave rise to internal hypersalivation, decrease in weight and acute motor ataxia. Detoxification was low and repeated administration therefore led to toxin accumulation. Response of the chicken to liver feeding was roughly quantitative; so liver, which is the most potential toxic tissue, may be used for a preventive screening test in ciguatera-endemic areas.

A study of the distribution of ciguatoxin in individual Caribbean fish.

Ciguatera toxins were extracted from the tissues of 36 poisonous fishes including 9 dangerous species collected in the Caribbean. Toxicity assays were carried out in mice and the distinctive symptoms of ciguatera poisoning were observed. In a single fish, ciguatoxin was found in the blood, flesh, gonads, gills, heart, skin and bones. The concentration was highest in the viscera and in particular in the liver, kidney and spleen. The ratios of the toxin concentrations of the liver or viscera to that of the flesh were high and varied with the species suggesting that the toxin is stored in different ways in different fish. Subcellular fractionation of liver cells revealed that most of the ciguatoxin was attached to cytoplasmic proteins and that some toxin was probably bound to the membranes.

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.

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.

[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.

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.

[Biological assay of ciguatoxin in the chicken: analysis of the symptoms induced and toxicity of extracts of ciguatoxic chickens of the island of Saint Barthelemy]. / Controle biologique de la ciguatoxine chez le poussin: analyse des symptômes induits et de la toxicité d'extraits de poissons ciguatoxiques de l'île de Saint-Barthélémy.

Ciguatera poisoning was studied in the chicken where it gave rise to internal hypersalivation, acute motor ataxia, low rectal temperature and arrested growth. LD50 figures indicated that the chicken is two to five time at least more sensitive to ciguatoxin than the mouse. So the chick is very convenient for laboratory studies in ciguatera research.

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.

[The absence of Sindbis virus from French Polynesia]. / A propos de l'absence du virus Sindbis en Polynésie française

The authors report, in French Polynesia that Sindbis antigen is a good control for rubella serodiagnosis. Data are presented on Hemagglutination Inhibition from 2,032 sera, showing no evidence of any Sindbis antigen circulation.

[Ciguatoxin and edible reef fishes]. / Ciguatoxine et poissons de récifs comestibles

Referring to the various human and animal clinical response to consumption of reef fish, the authors precise the importance of ingested dose and the role of cumulative effects. They point out the arbitrary character of distinguishing poisonous and edible fish of the same species. At the light of these data they find ciguatoxin in edible fish from known latent ciguateric potential species in atoxic areas. Therefore ciguatoxin does appear as a natural biotoxin permanently produced in most of the polynesian coral reefs ecosystems. The occurence of clinical disorders by men and animals results from a sudden increase of its production following biological transitory modification of some coral reef biota.