RESUMEN
Poor bioavailability and low residence time limit the efficiency of conventional biguanide-based eye drops against Acanthamoeba keratitis. The aim of this work was to formulate an original anti-amoebic thermoreversible ocular gel combining biguanide and metalloproteases inhibitor - chelating agent. Chlorhexidine digluconate (CHX)-ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were compounded in poloxamer 407 saline solution. 0.02% CHX - 0.1% Na2EDTA loaded thermosensitive ocular gel exhibited appropriate pH (5.73⯱â¯0.06), iso-osmolality (314⯱â¯5â¯mOsm/kg), viscosity (ranged between 15 and 25â¯mPa.s) and thermal gelation (26.5⯰C and 33⯰C) properties. Bioadhesion of gel was successfully tested onto isolated bovine eyes as well as the assessment of CHX penetration into the cornea. Intracorneal CHX concentration was found greater than trophozoite minimum amoebicidal concentration and minimal cysticidal concentration after 15-min and 2-h ocular exposure, respectively, while any CHX permeation through the cornea was detected (<51â¯ng/cm2/h). Improvement of CHX ocular bioavailability was attributed to probable solubilization of tear film lipid layer by poloxamer. In vitro efficiency of CHX-Na2EDTA ocular gel was confirmed from the drastic reduction of trophozoite and cyst survival (to 25% and 2%, respectively), confirming the potential of the multicomponent pharmaceutical material strategy for the treatment of Acanthamoeba keratitis.
Asunto(s)
Queratitis por Acanthamoeba/tratamiento farmacológico , Amebicidas/administración & dosificación , Clorhexidina/análogos & derivados , Ácido Edético/administración & dosificación , Administración Oftálmica , Amebicidas/farmacocinética , Amebicidas/farmacología , Animales , Disponibilidad Biológica , Bovinos , Quelantes/administración & dosificación , Quelantes/farmacocinética , Quelantes/farmacología , Química Farmacéutica/métodos , Clorhexidina/administración & dosificación , Clorhexidina/farmacocinética , Clorhexidina/farmacología , Córnea/metabolismo , Combinación de Medicamentos , Ácido Edético/farmacocinética , Ácido Edético/farmacología , Geles , Concentración Osmolar , Temperatura , Trofozoítos/efectos de los fármacos , ViscosidadRESUMEN
Free-living amoebae (FLA) are ubiquitous protozoa found worldwide in the environment. They feed by phagocytosis on various microorganisms. However, some bacteria, i.e., amoebae-resistant bacteria (ARB) or bacterial endocytobionts, can resist phagocytosis and even multiply inside FLA. This study investigated the diversity of culturable FLA in various soils from agricultural and mining sites and their bacterial endocytobionts. FLA were cultured on non-nutrient agar with alive Escherichia coli and identified by PCR and sequencing. Amoebae were lysed and bacterial endocytobionts were cultured on TSA 1/10 and Drigalski medium. Bacterial isolates were identified by PCR and 16S rDNA sequencing and characterized for their antibiotic resistance properties. To measure bacterial virulence, the amoebal model Dictyostelium discoideum was used. The analysis of FLA diversity showed that Tetramitus was the most prevalent genus in agricultural soil from Burkina Faso (73%) and garden soil from Vietnam (42%) while Naegleria and Acanthamoeba were dominant genera in mining soil from Vietnam (55%) and French alpine soil (77%). Some genera were only present in one out of the four soils analyzed. The bacterial endocytobiont included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Human opportunistic pathogens identified as Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia were found associated with amoebae including Micriamoeba, Tetramitus, Willaertia, or Acanthamoeba. Some of these bacteria showed various antibiotic resistance phenotypes and were virulent. Our study confirms that the occurrence of these opportunistic bacteria with FLA in soils may be important for the survival, multiplication, and spread of pathogens in the environment.
Asunto(s)
Acanthamoeba/microbiología , Amoeba/microbiología , Dictyostelium/microbiología , Escherichia coli/crecimiento & desarrollo , Naegleria/microbiología , Simbiosis/fisiología , Acanthamoeba/clasificación , Agricultura , Amoeba/clasificación , Burkina Faso , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Humanos , Naegleria/clasificación , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Suelo , Microbiología del Suelo , VietnamRESUMEN
An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Côte d'Azur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bacteria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers.
Asunto(s)
Diarrea/epidemiología , Diarrea/virología , Brotes de Enfermedades/estadística & datos numéricos , Norovirus/aislamiento & purificación , Carrera/estadística & datos numéricos , Virosis/epidemiología , Adolescente , Adulto , Trazado de Contacto/métodos , Trazado de Contacto/estadística & datos numéricos , Femenino , Francia/epidemiología , Juegos Recreacionales , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Factores de Riesgo , Virosis/virología , Adulto JovenRESUMEN
Naegleria sp. is a free living amoeba belonging to the Heterolobosea class. Over 40 species of Naegleria were identified and recovered worldwide in different habitats such as swimming pools, freshwater lakes, soil or dust. Among them, N. fowleri, is a human pathogen responsible for primary amoeboic meningoencephalitis (PAM). Around 300 cases were reported in 40 years worldwide but PAM is a fatal disease of the central nervous system with only 5% survival of infected patients. Since both pathogenic and non pathogenic species were encountered in the environment, detection and dispersal mode are crucial points in the fight against this pathogenic agent. Previous studies on identification and genotyping of N. fowleri strains were focused on RAPD analysis and on ITS sequencing and identified 5 variants: euro-american, south pacific, widespread, cattenom and chooz. Microsatellites are powerful markers in population genetics with broad spectrum of applications (such as paternity test, fingerprinting, genetic mapping or genetic structure analysis). They are characterized by a high degree of length polymorphism. The aim of this study was to genotype N. fowleri strains using microsatellites markers in order to track this population and to better understand its evolution. Six microsatellite loci and 47 strains from different geographical origins were used for this analysis. The microsatellite markers revealed a level of discrimination higher than any other marker used until now, enabling the identification of seven genetic groups, included in the five main genetic groups based on the previous RAPD and ITS analyses. This analysis also allowed us to go further in identifying private alleles highlighting intra-group variability. A better identification of the N. fowleri isolates could be done with this type of analysis and could allow a better tracking of the clinical and environmental N. fowleri strains.
Asunto(s)
Marcadores Genéticos , Repeticiones de Microsatélite , Naegleria fowleri/genética , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
Primary amoebic meningoencephalitis (PAM) is a fatal infection in most cases, caused by the amoeba flagellate Naegleria fowleri. This report describes the first cases of PAM in Algeria, in a cow and a ewe from Batna, north-eastern Algeria. The death of both ruminants occurred a week after the first clinical manifestations. The cerebrospinal fluid, after staining with May-Grünwald-Giemsa, showed the presence of amoebae cells. Histological sections revealed numerous amoebae in all parts of the brain. The presence of N. fowleri was confirmed using a species-specific real-time PCR in histological tissue sections. The two PAM cases were reported during the hot season, and the source of infection is very likely the water where the cattle came to drink. Particular attention should be focused on this type of infection in aquatic environments when the temperature is high and preventive measures must be taken to avoid the proliferation of N. fowleri.
Asunto(s)
Amebiasis/veterinaria , Enfermedades de los Bovinos/parasitología , Infecciones Protozoarias del Sistema Nervioso Central/veterinaria , Naegleria fowleri/aislamiento & purificación , Enfermedades de las Ovejas/parasitología , Argelia/epidemiología , Amebiasis/epidemiología , Amebiasis/parasitología , Amebiasis/transmisión , Animales , Bovinos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/transmisión , Infecciones Protozoarias del Sistema Nervioso Central/epidemiología , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , Infecciones Protozoarias del Sistema Nervioso Central/transmisión , Líquido Cefalorraquídeo/parasitología , Clima , ADN Protozoario/genética , Reservorios de Enfermedades , Femenino , Embarazo , Complicaciones Infecciosas del Embarazo/parasitología , Complicaciones Infecciosas del Embarazo/veterinaria , Estaciones del Año , Ovinos , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Ovejas/transmisión , Agua/parasitología , Abastecimiento de AguaRESUMEN
The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe.
Asunto(s)
Amebiasis/parasitología , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , ADN Protozoario/aislamiento & purificación , Naegleria fowleri/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , ADN Protozoario/química , Naegleria fowleri/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We examined a partial SSU-rDNA sequence from 20 Acanthamoeba isolates associated with keratitis infections. The phylogenetic tree inferred from this partial sequence allowed to assign isolates to genotypes. Among the 20 isolates examined, 16 were found to be of the T4 genotype, 2 were T3, 1 was a T5, and 1 was a T2, confirming the predominance of T4 in infections. However, the study highlighted other genotypes more rarely associated with infections, particularly the T2 genotype. Our study is the second one to detect that this genotype is associated with keratitis. Additionally, the phylogenetic analyses showed five main emerging clusters, T4/T3/T11, T2/T6, T10/T12/T14, T13/T16, and T7/T8/T9/T17, regularly obtained whichever method was used. A similar branching pattern was found when the full rDNA sequence was investigated.
Asunto(s)
Queratitis por Acanthamoeba/parasitología , Acanthamoeba/clasificación , Acanthamoeba/genética , Acanthamoeba/aislamiento & purificación , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Genotipo , Humanos , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
Investigation of soil amoebae in 11 cooling towers allowed us to isolate a major unknown small-sized amoeba population (SZA). However, SZA did not appear to be specific to cooling tower ecosystems since they are also a major amoeba population found in muds isolated from different points of a water treatment plant. The SSU-rDNA sequences from SZA strains did not match any known database sequences, suggesting that SZA constitutes a new amoeba taxon. We isolated and further described one of the SZA that we named Micriamoeba tesseris. The phylogenetic analyses showed that Micriamoeba tesseris belongs to the Amebozoa and branched together with genus Echinamoeba+Vermamoeba vermiformis. Phylogenetic analyses within the Micriamoeba group distinguished different subgroups of Micriamoeba strains according to their origin, i.e. cooling tower or mud. Although Micriamoeba are able to feed on viable E. coli cells, they do not uptake virulent Legionella pneumophila strains, thus enabling them to avoid infection by Legionella. Consequently, Micriamoeba is not directly involved in L. pneumophila multiplication. However, an indirect role of Micriamoeba in Legionella risk is discussed.
Asunto(s)
Amoeba/clasificación , Microbiología Ambiental , Amoeba/aislamiento & purificación , Amoeba/microbiología , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Escherichia coli/aislamiento & purificación , Conducta Alimentaria , Genes de ARNr , Legionella pneumophila/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
Its natural resistance to antiprotozoal chemotherapy characterizes the intestinal protozoan parasite Cryptosporidium parvum and the P-glycoprotein-related multidrug resistance proteins such as CpABC3 could be involved. In order to design and study specific inhibitors of the CpABC3 nucleotide-binding domain, a hexahistidine-tagged recombinant protein encompassing the N-terminal cytosolic NBD1 domain was overexpressed in E. coli and purified. The 45 kDa H6-NBD1 displayed intrinsic fluorescent properties consistent with the presence of two Trp residues in a hydrophobic environment. The binding of ATP and the fluorescent analogue TNP-ATP produced a dose-dependent quenching as well as progesterone and the flavone quercetin. The extrinsic fluorescence of TNP-ATP was enhanced upon binding to H6-NBD1, which was only partially displaced by the natural substrate ATP. The recombinant protein hydrolyzed ATP (K(m)=145.4+/-18.2 microM), but ADP (K(m)=4.3+/-0.6mM) and AMP (K(m)=5.4+/-1.5 microM) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (K(i)=36.6+/-4.5 microM), but quercetin and progesterone were not inhibitors, evidencing different binding sites. The recombinant C. parvum H6-NBD1 should be a valuable tool for rational drug design and will allow the discrimination between specific inhibitors of the catalytic site and molecules binding to other sites.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/aislamiento & purificación , Cryptosporidium parvum/enzimología , Cryptosporidium parvum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Progesterona/farmacología , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Quercetina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.