Caracterização da exposição humana a lodo de esgoto sanitário na cadeia: tratamento, uso agrícola e consumo de hortaliças / Characterization of human exposure to sewage sludge in the chain: treatment, agricultural use and consumption of vegetables

RESUMO Objetivou-se caracterizar a exposição humana à infecção por bactérias e vírus via ingestão, considerando os cenários: tratamento do lodo (cenário 1); uso do lodo como adubo agrícola (cenário 2) e consumo de hortaliças adubadas com lodo (cenário 3). A exposição foi caracterizada pelas concentrações de Escherichia coli, vírus entéricos cultiváveis (VEC) e colifagos somáticos (CS) nas amostras: lodo de esgoto em tratamento (LET), água de lavagem das mãos (ALM) no tratamento e no plantio, mistura solo + lodo (MSL) e hortaliças alface e cenoura. No cenário 1, em torno de 50-60 dias, as amostras LET apresentaram umidade < 10% e padrão classe A. Nas amostras LET, concentrações de CS variaram entre 9,1 × 102 e 1,9 × 105 UFP.g-1 ST e de VEC entre 2,42 e 7,15 UFP.g-1 ST. No cenário 2, CS foram detectados em 72% das amostras MSL com concentrações entre 10 e 330 UFP.mL-1. Concentrações de E. coli ≤ 102 NMP.100 mL-1 foram detectadas nas amostras ALM-tratamento (63,7%). Para vírus, todas as ALM-tratamento foram negativas e 17,4% das ALM-plantio apresentaram VEC entre 3 e 409,2 UFP.mL-1. Todas as amostras de alface foram negativas para CS e VEC. Considerando-se as características dos cenários avaliados, trabalhadores do serviço de saneamento, trabalhadores rurais e consumidores de hortaliças estiveram expostos a baixas concentrações de E. coli e VEC. CS foram resistentes ao tratamento térmico e ao plantio, o que sugere seu potencial como indicadores de VEC.

ABSTRACT The aim of this study was to characterize human exposure to microbiological contamination (bacteria and viruses) via ingestion, considering the following scenarios: treatment of sludge (scenario 1); use of sludge as fertilizer (scenario 2) and consumption of vegetables fertilized with sludge (scenario 3). Exposure was characterized by the levels of Escherichia coli, cultivable enteric viruses (CEV) and somatic coliphages (SC) in the samples: sewage sludge under treatment (SST); handwashing water (HW) in treatment and planting; mix soil + sludge (MSL) and the vegetables lettuce and carrots. In scenario 1, SST samples presented < 10% humidity and class A pattern around 50-60 days; SC concentrations ranged from 9.1 × 102 to 1.9 × 105 PFU.g-1 TS, and CEV ranged from 2.42 to 7.15 PFU.g-1 TS. In scenario 2, CS were detected in 72% of HW samples, and concentrations ranged from 10 to 330 PFU.mL-1. E. coli concentrations ≤ 102 NMP.100 mL-1 were detected in HW-treatment samples (63.7%). Viruses were not detected in 100% of HW-treatments samples, and 17.4% of HW-plantings had CEV concentrations between 3 and 409.2 PFU.mL-1. All the vegetable samples were negative for SC and CEV. Considering the characteristics of the scenarios evaluated, sewage workers, rural workers and consumers of vegetables were exposed to low concentrations of E. coli and CEV. Somatic coliphages were resistant to heat treatment and planting, which supports their potential as indicators of CEV.

Storage Quality of Functional Meat Nuggets with Biocontrol Films, Coliphages and Different Packaging Conditions

Functional spent hen meat nuggets (SMN) both unwrapped and wrapped in bioactive biodegradable films were evaluated for their storage stability at refrigeration temperature (4 ±1ºC) under different packaging conditions. Standardized zein films formulated with 5% zein proteins and 7% glycerol and incorporated with 5% coliphages having concentration of 1010 pfu/ml were utilized in this study. Different treatments such as T1 (product packaged in aerobic conditions), T2 (product packaged in modified atmospheric packaging [MAP] conditions), T3 (product packaged in vacuum), T4 (product wrapped in developed films and packaged in aerobic conditions), T5 (product wrapped in developed films and packaged in MAP conditions), T6 (product wrapped in developed films and packaged in vacuum conditions) were evaluated at weekly interval for 5 weeks for their physico-chemical, sensory and microbiological parameters. Results depicted that T5 (product wrapped in developed films and packaged in MAP conditions) had comparatively lower value for TBARS, Free fatty acids, peroxide value and standard plate count than other treatments. Psychrophiles, yeast and mold, E.coli, coliforms were completely absent in the T5 throughout the storage period. All vacuum and MAP packaged products had better sensory attributes throughout storage than aerobic packaged products. It was thus concluded that developed SMN can be successfully stored for 35 days in bioactive biodegradable wrap under MAP conditions at 4±1oC

Relación entre la presencia de colifagos en agua para consumo humano, las lluvias y las diarreas agudas en Costa Rica / Relation between coliphages presence in drinking water, rain and acute diarrhea in Costa Rica

ResumenLa calidad del agua de consumo es una de las preocupaciones más grandes a las que se enfrentan los entes de salud. En Costa Rica, a pesar de la buena calidad de las fuentes de agua y del agua de consumo, se siguen registrando numerosos casos de diarrea anualmente. En este estudio se realizó un análisis de indicadores de contaminación virales y bacterianos, en dos comunidades del Valle Central con acueducto propio. Se analizaron en total 24 muestras de agua por cada comunidad, durante un año, 12 en la fuente de agua y 12 en el agua tratada. La frecuencia de diarreas en cada comunidad fue comparada con la calidad microbiológica del agua consumida, encontrándose ausencia de indicadores bacterianos en muestras de agua tratada, mientras que la presencia de indicadores virales en el agua de consumo (con cloro residual) se relaciona con un incremento en el número de casos de diarreas, adicionalmente se analizó el comportamiento de las diarreas de acuerdo a la precipitación promedio con el fin de observar posibles patrones de estacionalidad. Estos hallazgos respaldan la necesidad de ampliar el monitoreo de calidad microbiológica del agua con otro tipo de indicadores de contaminación que correlacionen mejor con otros agentes patógenos de transmisión hídrica, principalmente en época seca, periodo en que se observan más casos de diarrea.

AbstractThe quality of potable water is one of the most important issues of public health. In Costa Rica, in spite of having good sources of water and good quality of water for consumption, there continues to be a significant number of diarrhea cases annually. In this investigation, viral and bacterial analyses of indicators of contamination were conducted in two rural communities with their own aqueducts in the Central Valley of Costa Rica. Twenty-four samples were taken from each community during the year; 12 from the water sources and 12 from the treated waters. The frequency of diarrhea in each community was compared to the microbiological quality of the water being consumed, noting an absence of bacterial indicators in samples of treated water, while the presence of viral indicators in the water (with residual chlorine) were associated with an incremental increase in the number of cases of diarrhea. The analysis also included measurements of average monthly rainfall with the aim of observing possible associations between seasonal conditions and the incidence of diarrhea cases. These findings indicate the need to increase the monitoring of the microbiological quality of the water with other types of indicators of contamination that correlate better with other pathogens transmitted by water, especially in the dry seasons when a greater number of cases of diarrhea are observed.

Avaliação da viabilidade de utilização de colifagos como indicadores de poluição fecal: suas relações com parâmetros físicos e químicos e indicadores bacterianos / Evaluation of the viability of using coliphages as indicators of fecal pollution: relations with physical and chemical parameters and bacterial indicators

RESUMO As bactérias coliformes, correntemente utilizadas como indicadores de contaminação fecal, não asseguram a inexistência de outros microrganismos patogênicos em águas, principalmente vírus entéricos. O objetivo do trabalho foi avaliar a viabilidade da utilização de colifagos como indicadores virais no efluente de estações de tratamento de esgotos (ETE), pois sua análise apresenta um método mais simplificado quando comparado aos procedimentos para identificação de vírus entéricos. O comportamento de colifagos nas diferentes estações climáticas durante o período de um ano foi verificado no efluente final da ETE, apresentando-se em maior quantidade nos períodos secos. Não foi verificada relação significativa entre colifagos e coliformes, bem como com as variáveis demanda bioquímica de oxigênio (DBO5), turbidez e sólidos totais. Os resultados obtidos nesta pesquisa indicam a necessidade do monitoramento conjunto de vírus e bactérias, a fim de garantir adequada avaliação da qualidade do esgoto tratado.

ABSTRACT Coliform bacteria, currently used as fecal indicator, does not ensure the absence of other pathogenic microorganisms in water, mainly enteric viruses. The objective of this study was to evaluate the viability of using coliphages as viral indicators in the effluent from Wastewater Treatment Plants (WWTP) due to the fact that Coliphages determination involves a simplified methodology. The seasonal pattern of coliphages was measured in the final effluent of a domestic WWTP, which occurred in higher values, mainly during dry seasons periods. This behavior was similar with the described by the authors in relation to enteric viruses in water. Significant relationship was not found among coliphages and coliforms, as well as with the physical and chemical parameters analyzed. The need for joint monitoring of viruses and bacteria is emphasized, in order to ensure the proper quality of treated water.

Metodología rápida y sencilla para la determinación de colifagos somáticos como indicadores de contaminación fecal en una planta de tratamiento de agua localizada al noreste colombiano / Quick and easy methodology to determine somatic coliphages as indicators of fecal contamination in a water treatment plant located in northeastern Colombia

Introducción: En el presente artículo se propone una metodología rápida y sencilla para determinar colifagos sómaticos en las diferentes etapas del tratamiento de agua de una planta del Nordeste Colombiano, con el fin de verificar la calidad del proceso de potabilización. Materiales y métodos: Se empleó la técnica de filtración por membrana para obtener el contenido fágico y posteriormente se cuantificó la presencia de los virus a través de la metodología de capa simple usando agar tripticasa de soya modificado. Resultados: El 88% de las muestras de agua analizadas resultaron ser positivas en alguna de las etapas del proceso de potabilización. La presencia de fagos aumentó en época de lluvias con recuentos máximos de 1310 UFP/100mL en la etapa de captación del agua y de 2,5 UFP/100mL en la etapa de cloración. La prueba no-paramétrica Chi cuadrado de homogeneidad en distribución corroboró estadísticamente la igualdad en distribución de las muestras positivas encontradas en cada una de las etapas de potabilización (p>5%) pues el muestreo fue fijado por indicador. Conclusión: La metodología empleada permitió en tan solo 12 horas determinar la presencia de colifagos somáticos en las diferentes etapas de tratamiento de agua de la planta objeto de estudio. Además, se comprobó la elevada resistencia de estas nanomáquinas naturales a los procesos de desinfección, que mostraron persistencia en etapas de almacenamiento y distribución, especialmente en época de lluvias.

Introduction: In this article a quick and simple method is proposed to determine somatic coliphages in the different stages of water treatment in a plant in the northeast of Colombia, in order to verify the quality of the treatment process. Materials and methods: The technique of membrane filtration was used to get the phage content and subsequently through the methodology of single layer using tryptic soy agar modified, the presence of the virus was quantified. Results: 88% of the water samples analyzed were found to be positive in any of the stages of purification. The presence of phages increased during the rainy season with maximum counts of 1310 UFP / 100 mL at the stage of uptake of water and 2.5 UFP / 100 mL in the chlorination stage. Non-parampetrica Chi square homogeneous distribution test confirmed statistically equal distribution of positive samples found in each of the stages of purification (p> 5%) because the sample was determined by indicator. Conclusion: The methodology allowed to determine the presence of somatic coliphages at different stages of water treatment in the plant under study in only 12 hours. In addition, the high resistance of these natural nanomachines to the processes of disinfection was found, which showed persistence in warehousing and distribution stages, especially in the rainy season.

Evaluación de l calidad microbiológica del agu para consumo y del agua residuual en una población de bogotá (Colombaa) / Evaluatin of thee microbial quality of dinking water and wastewater in one population of bogota (Colombia)

En Colombia el acceso al agua potable y a sistemas de saneamiento no están garantizados para buena parte de la población, por lo que el consumo de agua contaminada genera enfermedades de origen hídrico. La alternativa más utilizada para el acceso al agua potable es su almacenamiento dentro de las viviendas y la improvisación de sistemas de alcantarillado que generalmente se encuentran a cielo abierto. Objetivo: Evaluar la calidad microbiológica del agua utilizada para consumo y el agua residual cercana a las viviendas de una población en desplazamiento y su relación con la salud de la población. Materiales y Métodos: Se recolectaron 36 muestras de agua para consumo almacenadas en diferentes tipos de recipientes y de grifos, en caso de estar instalados. Adicionalmente se recolectaron 15 muestras de agua residual de forma aleatoria en un asentamiento ilegal compuesto en su gran mayoría por población desplazada. Para evaluar la calidad microbiológica del agua se analizaron como indicadores de contaminación de origen fecal E. coli, C. perfringens y colífagos somáticos. Simultáneamente se llevó a cabo la aplicación de encuestas de morbilidad sentida con el fin de establecer la posible relación entre las condiciones de vida de los habitantes, la calidad del agua y la percepción del estado de salud. Resultados y Conclusiones: En 14 de las 36 casas seleccionadas y en las 15 de redes de alcantarillado a cielo abierto, se encontró contaminación de origen fecal. La encuesta de morbilidad realizada muestra cómo la calidad del agua y las condiciones de vida pueden influir en la salud de la población. La OMS considera que el agua para consumo debe estar libre de microorganismos que afecten la salud humana, para lo cual es importante su tratamiento, almacenamiento y aislamiento de redes de aguas residuales.

In Colombia access to drinking water and sanitation are not guaranteed for the majority of the population, reason why the consumption of contaminated water generates water-borne diseases. The most commonly used alternative to access drinking water is its storage in homes and the improvisation of sewerage systems typically found outdoors. Objective: To evaluate microbiological quality of drinking water and wastewater near the homes of displaced populations and its relationship with the community's health. Materials and Methods: Thirty-six (36) samples of drinking water for consumption stored in different types of containers and taps were collected where these were installed. Additionally 15 samples of wastewater were collected randomly in an illegal settlement, where most of the inhabitants were displaced persons. E. coli, C. perfringens and somatic coliphages were examined as fecal contamination indicators for microbiological water quality assessment. Simultaneously, morbidity surveys were carried out to establish the possible relationship between the inhabitants' living conditions, water quality and health status perception. Results and Conclusions: Contamination of fecal origin was found in 14 out of the 36 selected houses and in 15 outdoors sewerages. The conducted morbidity survey shows how water quality and living conditions can influence the health of the population. WHO estimates that drinking water should be free from microorganisms that affect human health, reason why water treatment, proper storage and isolation of residual water are important facts to be considered.

Detección de virus entéricos en un sistema piloto de tratamiento de aguas residuales municipales / Detection of Enteric Viruses in a Pilot System for Municipal Wastewater Treatment

Se evaluó un sistema piloto de tratamiento de aguas residuales municipales para verificar su capacidad de remoción de patógenos virales. La presencia de Adenovirus, Calicivirus, Enterovirus, Virus de Hepatitis A y Virus de Hepatitis E. Se determinó mediante RT-PCR (Reacción en Cadena de la Polimerasa con Transcriptasa Reversa). Además se examinó la eficacia de los colifagos como indicadores y se verificó la presencia de indicadores bacterianos clásicos. Se obtuvo un 100% de positividad para la presencia de todos los virus en la entrada y salida del sistema, por lo que el sistema no es capaz de eliminarlos. Los colifagos no fueron removidos por este sistema, pero sirvieron como indicadores para la presencia de virus entéricos en el mismo. Los indicadores bacterianos redujeron su número a su paso por el sistema, pero aún al final del sistema, los coliformes se encontraban en números por encima de los límites establecidos por las leyes venezolanas. Por lo tanto, sería necesario agregar otros pasos al sistema, para poder obtener un efluente que cumpla las leyes y que garantice la ausencia de virus entéricos.

A pilot system for treating municipal waste was evaluated to verify its ability to remove viral pathogens. The presence of Adenovirus, Calicivirus, Enterovirus and Hepatitis A and E was determined using RT-PCR. The efficiency of coliphages as indicators and the presence of classic bacterial indicators were also investigated. A 100% positivity was obtained for the presence of all viruses at the entrance and exit of the system; therefore, the system was not able to remove them. Coliphages weren’t removed by the system, but they were effective as indicators of enteric viral presence in it. Bacterial indicators reduced their number going through the system, but even at the end of the system, coliforms had numbers above the limits dictated by Venezuelan law. Therefore, it would be necessary to add additional steps to the system in order to obtain an effluent that meets the laws and to ensure the absence of enteric viruses.

Implementación de dos métodos de recuento en placa para la detección de colifagos somáticos, aportes a las metodologías estándar

Los métodos de recuento en placa capa doble y capa simple de agar, para la cuantificación de colifagos somáticos en aguas, fueron implementados utilizando como base metodologías estándar. Diferentes variables fueron ensayadas, lo cual permitió la precisión en algunos pasos no incluidos en metodologías estándares. De los hallazgos de mayor importancia, se exponen las consecuencias de utilizar un cultivo de Escherichia coli excesivamente concentrado y se describe la obtención de un cultivo en fase logarítmica en solo 4 horas de incubación, ajustando la concentración a una densidad óptica de 0,3 a 600nm (3,1 x 10(8)UFC/ mL), o a un McFarland 1 (3,0 x10(8)UFC/ mL). Se determinó que los controles de colifagos deben ser almacenados a -70 °C para reducir su degradación y que se deben evitar cantidades superiores a 20 mL de mezcla de reacción por plato de Petri, para reducir las burbujas que pueden interferir con la lectura de unidades formadoras de placas (UFP). Se demostró que los colifagos de las muestras de agua pueden almacenarse 48 horas a 4 °C sin que sufran degradación y que en las muestras con altas concentraciones de colifagos no se observa UFP porque se da una lisis confluente de la capa bacteriana. No se encontraron diferencias significativas en la recuperación de colifagos al utilizar un método u otro, pero dichos métodos deben ser evaluados por medio de controles, antes de aplicarlos directamente en el análisis de muestras de agua.

Two plate count methods, double layer and single layer of agar for quantification of somatic coliphages in water, were implemented using standard methodologies. Several variables were tested and provided valuable information that was not included in standard methodologies. The most important findings are described, such as the effect of using an excessively concentrated culture of E. coli and production of a log phase culture in only 4 hours of incubation, adjusting the concentration to an optical density of 0.3 at 600 nm (3.1 x 108 CFU / mL), or to McFarland 1 (3.0 x 108 CFU / mL). It was determined that coliphages controls must be stored at -70 °C to reduce its degradation. Quantities of reaction mixture exceeding 20 mL per Petri dish must be avoided to prevent interfere with bubbles during the counting of plate forming units (PFU). It was demonstrated that coliphages isolated from water samples can be stored for 48 hours at 4 °C without any degradation, and PFU are not observed in samples with high concentrations of coliphages, because a confluent lysis of the bacterial layer. There was no significant difference in the recovery of coliphages using doble layer or single layer methods, but such methods should be evaluated by means of controls, before applying them directly in the analysis of water samples.

Pesquisa de bacteriófagos em água suspeita de contaminação por vírus da hepatite A / Study of bacteriophages in water suspected of contamination by hepatitis A virus

Os vírus entéricos, quando presentes no trato gastrointestinal de indivíduos infectados, são eliminados pelas fezes em grandes quantidades podendo contaminar, direta ou indiretamente, as águas destinadas ao consumo humano. O ser humano é o único hospedeiro natural do vírus da hepatite A. A detecção de colifago e de fago de Shigella sonnei em água pode ser indicativa da presença destes vírus. No presente estudo foi realizado o diagnóstico laboratorial presuntivo de um surto de hepatite A, pela pesquisa de bacteriófagos, em uma amostra de água de poço e o estudo da sobrevivência dos fagos de S. sonnei em água. A análise revelou resultado positivo para fagos de Shigella e negativo para colifagos. Quanto aos ensaios de sobrevivência, a presença dos fagos de S. sonnei foi detectada na água do poço naturalmente contaminada até o nono dia de conservação em temperatura ambiente.

The enteric viruses are present in the gastrointestinal tract of infected individuals, being eliminated in feces in large quantities and can directly or indirectly contaminate water intended for human consumption. Humans are the only natural hosts for hepatitis A. Detection of coliphages and phage of Shigella sonnei in water may indicate the presence of these viruses. In the present study was done the presumptive laboratory diagnosis of an outbreak of hepatitis A through search of bacteriophages in a sample of well water and the study of survival of shigelafagos in water. The analysis revealed positive for phages of S. sonnei and negative for coliphages. For the tests of survival, the presence of phages of S. sonnei was detected in naturally contaminated well water until the ninth day of storage at room temperature.

Detection of somatic coliphages as indicators of faecal contamination in estuarine waters / Detección de colifagos somáticos como indicadores de contaminación fecal en aguas estuarinas

The appearance of enteric disease outbreaks associated with the use of waters considered bacteriologically suitable, calls for the search of new and more precise indicators. Samples of estuarine water were collected in order to quantify E. coli and E. coli ATCC 13706 somatic coliphages and to compare the usefulness of the latter to detect faecal contamination when the concentration of traditional indicators is not quantifiable. Statistical analyses suggested the division of sampling sites into two groups: group I and group II, according to the minor or major level of faecal pollution respectively registered. In group II a high correlation between the coliphages and E. coli (r: 0.73 p<0.01) was detected. E. coli always exceeded coliphage abundance. In group I, this relationship was statistically significant (r: 0.55 p< 0.05), coliphage counts were higher than those of E. coli and were detected in the absence of the latter. In summary, the use of E. coli ATCC 13706 somatic coliphages is proposed as a complementary tool for the diagnosis of the level of faecal contamination of estuarine waters, especially in areas of low pollution.

La aparición de brotes de enfermedades víricas entéricas asociadas al uso de aguas bacteriológicamente aptas impone la búsqueda de nuevos y más precisos indicadores de contaminación. Se recolectaron muestras de agua estuarina, donde se cuantificaron simultáneamente la bacteria E. coli y los colifagos somáticos de E. coli ATCC 13706, a fin de evaluar la utilidad de estos últimos para detectar contaminación fecal cuando la concentración de los indicadores tradicionales no es cuantificable. Los resultados estadísticos sugirieron la división de las estaciones de muestreo en dos grupos, I y II, de acuerdo con el menor o mayor nivel de contaminación fecal registrado, respectivamente. En el grupo II se detectó una alta correlación entre los recuentos de colifagos y de E. coli (r: 0,73 p<0,01). Asimismo, en este grupo la abundancia de E. coli siempre superó a la de colifagos. En el grupo I la correlación fue estadísticamente significativa (r: 0,55 p < 0,05), pero de mediana magnitud, los recuentos de colifagos superaron a los de E. coli, y éstos fueron detectados aun en ausencia de la bacteria. En conclusión, los colifagos somáticos de E. coli ATCC 13706 serían una herramienta accesoria en el diagnóstico del nivel de contaminación fecal de aguas estuarinas, sobre todo en áreas donde ésta es baja.