Aislamiento y caracterización de coliformes injuriados provenientes de la red de distribución de agua de bebida de La Plata, Argentina / Isolation and characterization of injured coliforms from the drinking water distribution network of La Plata, Argentina

We screened the La Plata drinking water distribution network for fecal and total coliform bacterial indicator by purification procedures, cultivating 66 membrane-filtered samples from the two networks on m-T7 agar. Subterranean and river-derived water yielded 13 and 18 confirmed gram-negative bacillus isolates, with 54 and 72 representing total coliforms, respectively. Those from the former source were Klebsiella oxytoca, Enterobacter agglomerans, and Enterobacter aerogenes and from the latter Klebsiella oxytoca, Enterobacter agglomerans, and Enterobacter cloacae, genomic group 3. Since 58 of the samples were positive using m-T7 medium it is suggested that the inclusion in standard quality control protocols should be implemented.

Relación entre el gradiente discontinuo de sacarosa y la viabilidad de ooquistes de Cryptosporidium sp / Relationship between discontinuous sucrose gradient and viability of Cryptosporidium sp. oocysts

Cryptosporidium sp. causes self-limiting gastroenteritis in immunocompetent individuals and can produce a life-threatening chronic diarrhea in immunodeficient patients. In order to obtain populations of selectively infective oocysts for inoculation in biological experiments, we developed an operational protocol for the enrichment of viable oocysts from crude fecal material. Using either fresh or formaldehyde-fixed feces as sources of viable and nonviable oocysts, respectively, we fractionated the samples on parallel discontinuous sucrose gradients and evaluated oocyst viability at different banding densities by an in vitro excystation assay. The formaldehyde-inactivated fecal samples formed no bands after centrifugation and 91.66 of the oocysts became concentrated in the pellet. Fresh fecal samples formed three bands at densities 1.062, 1.092, and 1.121, in addition to a sediment. Here the viability of the gradient-sedimented oocysts was 92.3 overall, and of those in the second band 100. Modifications in oocyst permeability thus seems to alter their sedimentation characteristics so that consequent distribution on sucrose gradients can be correlated with oocyst viability. Discontinuous sucrose density gradient sedimentation would thus constitute a simple and rapid mean to obtain viable oocysts for use in biological models both in vivo and in vitro.

Factores que influyen en el desenquistamiento in vitro de Cryptosporidium sp / Factors affecting the in vitro excystation of Cryptosporidium sp

Para evaluar la viabilidad de los ooquistes de Cryptosporidium sp se desarrolló y optimizó una técnica de desenquistamiento in vitro. Los ooquistes de Cryptosporidium sp se obtuvieron de una muestra de materia fecal de un niño con cryptosporidiosis sintomática. La muestra fue procesada por la técnica de gradiente discontinuo de sacarosa hasta obtener una suspensión de ooquistes purificados y concentrados (OPC). Los ooquistes se cuantificaron en cámara de Neubauer y preservaron en PBS pH 7, 2 a 4§C. Los dos protocolos ensayados a partir de alícuotas de 100µl de OPC con 178.000 ooquistes/mm3 fueron: I. Tratamiento con bilis 1 por ciento y bicarbonato de sodio al 0,44 por ciento a 37§C durante 24 horas. II Tratamiento con bilis 1 por ciento con pH 6 y pH 8, con y sin bicarbonato de sodio al 0,44 por ciento, con y sin incubación en atmósfera de 10 por ciento de CO2 durante 24 h a 37§C. Se evaluó el grado de desenquistamiento en todos los grupos efectuando el conteo de ooquistes intactos en cámara de Neubauer. La preincubación con tripsina no incrementó el desenquistamiento de los ooquistes de Cryptosporidium sp. Las mejores condiciones de desenquistamiento fueron utilizando bilis al 1 por ciento pH 8, incubado con 10 por ciento de CO2, a 37§C durante 24 h.