The free amoeba pathogens of warm waters of Guadeloupe. : ecological study, molecular characterization and prophylaxis of bathing areas.

Les amibes sont des organismes unicellulaires eucaryotes, de 10 à 300 µm, pouvant vivre sous forme parasite ou évoluer librement dans les sols et les milieux aquatiques ou encore adopter l’une ou l’autre forme en fonction de l’environnement. Certaines amibes libres sont hautement pathogènes pour les animaux et l’homme, en particulier Naegleria fowleri qui se développe dans les eaux naturellement chaudes à des températures comprises entre 27 et 45°C. Cette espèce est responsable d'une encéphalite généralement mortelle, la méningo-encéphalite amibienne primitive (MEAP), dont un cas a été recensé en Guadeloupe en 2008. Un enfant de 9 ans ayant contracté la maladie est décédé quelques jours après un bain dans les eaux chaudes de Dolé, sur la commune de Gourbeyre. Notre travail de Thèse à l’Institut Pasteur, en collaboration avec l’ARS, a permis d’initier un programme de recherche sur ces amibes pathogènes très peu connues sur notre territoire, afin de mieux évaluer le risque lié aux baignades dans les sources géothermales de la Guadeloupe. Nous avons développé et mis au point une méthode de détection et de dénombrement des amibes faisant appel à la biologie moléculaire qui est aujourd’hui appliquée en routine au sein de notre laboratoire. Depuis Janvier 2011, un suivi mensuel a révélé la présence des amibes thermophiles et de l’amibe pathogène dans presque tous les sites analysés (Ravine Chaude, Bains de La Lise et Bain du Curé à Pigeon, Bains Jaunes, Bain de Dolé, Bain de Capes, Bain des Amours, Bain de la rivière Grosse-Corde, Chute du Carbet, Bain de Morphy et Bain chaud de Matouba), à l’exception des eaux chaudes soufrées et/ou salées de Sofaïa et de l’Anse Thomas à Bouillante. Le séquençage des produits de PCR obtenus et déposés dans Genbank nous a permis de confirmer l’appartenance de cette souche de N. fowleri, au type 3 Euro-américain, identique à celui détecté lors de l’accident de 2008 (Moussa et al. 2013). Le nombre d’amibes pathogènes retrouvées varie de 2 à 30 amibes/litre selon les bains, sans dépasser la limite de 100 amibes/litre recommandée par les autorités sanitaires. En dépit de ces faibles concentrations, une surveillance régulière des bassins est nécessaire. Sur l’ensemble de l’année 2011-2012, l’espèce pathogène N. fowleri a été la plus fréquemment rencontrée par rapport aux espèces non pathogènes N. lovaniensis et Hartmanella sp. Ces données de surveillance ont amené l’ARS et les communes concernées à organiser une campagne de sensibilisation à destination des populations locales et touristiques, reposant sur l’installation de panneaux de prévention à proximité des principaux sites où N. fowleri a été retrouvée.La présence régulière de N. fowleri tout au long de l’année dans la plupart des bains chauds et notamment dans les bains les plus fréquentés, a orienté notre travail sur la recherche de l’origine de leur contamination. Nous avons découvert que les sources géothermales ne sont pas contaminées à leur émergence, mais que les amibes proviennent de la terre en amont des bassins. Ce résultat qui montre que le sol est le réservoir naturel des amibes, nous a permis de proposer aux collectivités un moyen de lutte efficace en canalisant l’eau depuis l’émergence jusqu’au bassin, sans passer par le sol.Amoebas are eukaryotic unicellular organisms, measuring from 10 to 300 µm, being able to live under a parasite form or to evolve freely in soils and aquatic media. They can even adopt either form depending of the environment. Some free living amoebas are highly pathogenic for animals and humans, especially Naegleria fowleri which develops in naturally hot waters at temperatures between 27 and 45°C. This species is responsible for an encephalitis generally lethal, the primary amoebic meningoencephalitis (PAM), a case of which occured in Guadeloupe in 2008. A 9-year-old child having contracted the disease, died a few days after a bath in the hot waters of Dolé, in Gourbeyre. Our Thesis at the Institut Pasteur of Guadeloupe, in association with the ARS, initiated a research program on these pathogenic amoebas poorly known in our territory, to better estimate the risk to contract the disease while bathing in geothermal recreational waters of Guadeloupe. We developed and worked out a method of detection and enumeration of amoebas using the molecular biology, which is currently applied in routine in our laboratory. Since January, 2011, a monthly monitoring of the baths revealed the presence of thermophilic free living amoebas and pathogenic amoeba in almost all the analyzed sites (Ravine Chaude, La Lise, Bain du Curé à Pigeon, Bains Jaunes, Bain de Dolé, Bain de Capès, Bain des Amours, Bain de la rivière Grosse-Corde, Chutes du Carbet, Morphy and the hot bath of Matouba), with the exception of sulphurated and/or salty hot waters of Sofaïa and the Anse-Thomas in Bouillante. The sequencing of the PCR products obtained were deposited in Genbank and confirmed that the Guadeloupean N. fowleri belonged to the Type-3 Euro-American, identical to that detected in the patient in 2008 (Moussa et al. 2013). The number of pathogenic amoebas varied from 2 to 30 amoebas / liter according to the baths, without exceeding the limit of 100 amoebas / liter recommended by the health authorities. In spite of this rather low concentration the baths require a regular surveillance. On the full year 2011-2012, the pathogenic species N. fowleri was the most frequently encountered species followed by N. lovaniensis and Hartmanella sp. These data of the surveillance led the ARS and the municipalities concerned to organize an awareness campaign destinated to the local and tourist population, based on the installation of prevention panels near the main sites where N. fowleri was found. The regular presence of N. fowleri during the year in most of the hot baths, especially the most frequented ones, leads us to look for the origin of their contamination. We discovered that geothermal springs are not contaminated at their emergence, but that amoebas come from the soil upstream the baths. This result which shows that soil is the natural reservoir of amoebas, allowed us to propose to the authorities effective means of prevention by installing pipes to carry the water from the emergence to the bath, without touching the soil

An Optimized Most Probable Number (MPN) Method to Assess the Number of Thermophilic Free-Living Amoebae (FLA) in Water Samples

International audienceDetection and quantification of pathogenic free-living amoebae (FLA) in water samples is critical for assessing water quality and for disease management issues. The most probable number (MPN) is commonly used to account for FLA in water. Nevertheless, this requires a high number of water replicates and working volumes, and a consequent number of non-nutrient agar (NNA)-plates seeded with Escherichia coli. Herein, we aimed at optimizing this difficult method, taking also into account key factors such as (i) the counting method, (ii) the delay between sample collection and sample processing, and (iii) the temperature during water sample transportation. To simplify the MPN method, we filtrated 1 × 1000 and 1 × 100 mL water samples, and cellulose acetate filters were cut in 10 parts and inverted on NNA-plates overlaid with E. coli. The comparison between the classical and our optimized MPN method showed that the final counts were similar, therefore validating the use of the optimized method. Our results also showed that for thermophilic FLA (such as Naegleria fowleri), water samples can be kept at around +30 • C and processed within 24 h. This improved MPN method is now routinely used in our laboratory to control Naegleria sp. in the water samples in Guadeloupe

Soil is the origin for the presence of Naegleria fowleri in the thermal recreational waters

International audienceNaegleria fowleri is found in most geothermal baths of Guadeloupe and has been responsible for the death of a 9-year-old boy who swam in one of these baths in 2008. We wanted to determine the origin for the presence of this amoeba in the water.Water samples were taken at the origin of the geothermal sources and at the arrival in the baths. After filtration, cultures were made and the number of Naegleria present was determined using the most probable number method. Soil samples collected in the proximity of the baths were also tested for the presence of thermophilic amoebae. The species identification was obtained by PCR. During three consecutivemonths, no Naegleria could be found at the origin of any geothermal source tested. In contrast, N. fowleri was isolated at least once in all baths at the arrival of the water, except one. Thermophilic amoebae could be found in each soil sample, especially near the baths located at a lower altitude, but N. fowleri was only isolated near two baths, which were also the baths most often contaminated with this species. So it appears that the contamination of the water with N. fowleri occurs after emerging from the geothermal source when the water runs over the soil. Therefore, it should be possible to reduce the concentration of N. fowleri in thegeothermal baths of Guadeloupe to for example less than 1 N. fowleri/10 L by installing a pipeline between the geothermal sources and the baths and by preventing flooding water from entering the baths after rainfall. By taking these measures, we were able to eliminate N. fowleri from a poollocated inside a reeducation clinic

Survey of Naegleria fowleri in Geothermal Recreational Waters of Guadeloupe (French West Indies)

In 2008 a fatal case of primary amoebic meningoencephalitis, due to the amoeboflagellate Naegleria fowleri, occurred in Guadeloupe, French West Indies, after a child swam in a bath fed with geothermal water. In order to improve the knowledge on free-living amoebae in this tropical part of France, we investigated on a monthly basis, the presence of Naegleria spp. in the recreational baths, and stream waters which feed them. A total of 73 water samples, 48 sediments and 54 swabs samples were collected from 6 sampling points between June 2011 and July 2012. The water samples were filtered and the filters transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli while sediment and swab samples were placed directly on these plates. The plates were incubated at 44uC for the selective isolation of thermophilic Naegleria. To identify the Naegleria isolates the internal transcribed spacers, including the 5.8S rDNA, were amplified by polymerase chain reaction and the sequence of the PCR products was determined. Thermophilic amoebae were present at nearly all collection sites. The pathogenic N. fowleri was the most frequently encountered thermophilic species followed by N. lovaniensis. The concentration of N. fowleri was rather low in most water samples, ranging from 0 to 22 per liter. Sequencing revealed that all N. fowleri isolates belonged to a common Euro-American genotype, the same as detected in the human case in Guadeloupe. These investigations need to be continued in order to counsel the health authorities about prevention measures, because these recreational thermal baths are used daily by loca

Vibrio aestuarianus subsp. cardii subsp. nov., pathogenic to the edible cockles Cerastoderma edule in France, and establishment of Vibrio aestuarianus subsp. aestuarianus subsp. nov. and Vibrio aestuarianus subsp. francensis subsp. nov.

Cockle mortality events have been reported in northern France since 2012. In the present study, we describe and investigate the implication of a potential bacterial causative agent in cockle mortality. Bacteria isolated from five different cockle mortality events were characterized and studied. Using phenotypic analysis combined with DNA–DNA hybridization (DDH) and whole genome sequencing, the isolates were shown to belong to Vibrio aestuarianus , a species regularly detected in France during oyster mortality events. Comparison of the strains from cockles with strains from French oysters and the type strain showed that the strains from cockles were genetically different to those from oysters and also different to the V. aestuarianus type strain. Moreover, the cockle and oyster strains were classified into two different, but close, groups both separated from the type strain by: (1) analyses of the ldh gene sequences; (2) DDH assays between 12/122 3T3T (LMG 31436T=DSM 109723T), a representative cockle strain, 02/041T (CIP 109791T=LMG 24517T) representative oyster strain and V. aestuarianus type strain LMG 7909T; (3) average nucleotide identity values calculated on the genomes; and (4) phenotypic traits. Finally, results of MALDI-TOF analyses also revealed specific peaks discriminating the three representative strains. The toxicity of representative strains of these cockle isolates was demonstrated by experimental infection of hatchery-produced cockles. The data therefore allow us to propose two novel subspecies of Vibrio aestuarianus : Vibrio aestuarianus subsp. cardii subsp. nov. for the cockle strains and Vibrio aestuarianus subsp. francensis subsp. nov. for the Pacific oyster strains, in addition to an emended description of the species Vibrio aestuarianus

PCR results for the three major thermophilic amoeba species found in Guadeloupe.

<p>PCR detection and agarose gel electrophoresis of ITS rDNA of <i>N. fowleri</i> (A), <i>N. lovaniensis</i> (B) and <i>Hartmannella</i> sp<b>.</b> (C). <b>M :</b> 100 bp ladder, <b>PC</b> : Positive control (DNA of <i>Naegleria fowleri</i> (#. 359)) gives a single PCR product of 359 bp with the NFITS primers and a 448 bp with the ITS primer set. <b><i>N.f</i></b> : <i>Naegleria fowleri, </i><b><i>N.l</i></b> : <i>Naegleria lovaniensis</i>, the ITS amplification gives a 403 bp PCR product while no band was obtained with the NFITS primers, <b><i>Hart</i></b><i>.</i> : <i>Hartmannella</i> sp., the ITS amplification gives a 900 bp while no band was observed with the NFITS primer set.</p

Distribution of sequenced isolates from the different geothermal baths of Guadeloupe.

<p>Distribution of sequenced isolates from the different geothermal baths of Guadeloupe.</p

Survey of free-living amoeba isolated from geothermal recreational waters of Guadeloupe on a montly basis (2011).

<p><b>Sp. :</b> thermophilic amoebae non-Naegleria sp., <b>N.l</b> : Naegleria lovaniensis., <b>N.f</b> : Naegleria fowleri,</p><p>(n) number of amoebae per liter of water; (+ ou -) presence or absence in sediment or swab; (blank) not investigated.</p

ITS1 sequences of the three isolates identified in the different geothermal waters of Guadeloupe.

<p>ITS1 sequences of the three isolates identified in the different geothermal waters of Guadeloupe.</p

Number of <i>Naegleria fowleri</i> isolated from the most frequented baths of Guadeloupe according to temperature and pH (2011–12).

<p>ND : non determined.</p