Comparação de métodos para quantificação de bactérias nitrificantes / Comparison of methods for quantification of nitrifying bacteria

RESUMO A quantificação de bactérias nitrificantes é de extrema importância para o monitoramento de sistemas biológicos de tratamento que promovam a nitrificação. Neste trabalho, 15 amostras de efluentes coletadas em sistema de tratamento por lodos ativados (LA) foram analisadas de modo a quantificar bactérias nitrificantes por meio de duas técnicas: tubos múltiplos ou técnica do número mais provável (NMP); e hibridação in situ fluorescente (FISH). Os resultados sugerem que houve uma tendência de se obter valores diferentes para bactérias oxidadoras de amônia por meio da NMP em comparação com a FISH. Não obstante, a análise estatística revelou que a diferença de quantificação encontrada entre as técnicas não foi significativa, indicando que ambas podem ser usadas. Para as oxidadoras de nitrito, não foi possível realizar comparação, uma vez que os gêneros que estavam sendo determinados em cada uma das técnicas provavelmente eram diferentes. Sendo assim, as técnicas NMP e FISH se mostraram métodos relativamente simples e adequados para quantificação de microrganismos nitrificantes, com vantagens e limitações inerentes a cada uma.

ABSTRACT The quantification of nitrifying bacteria is of utmost importance for monitoring biological treatment systems designed to promote nitrification. In this study, 15 activated sludge samples were analyzed in order to quantify nitrifying bacteria by two different methods: the most-probable number (MPN); and the fluorescence in situ hybridization (FISH). The results suggest that there was a tendency to obtain different values for ammonia-oxidizing bacteria by MPN compared to FISH. However, statistical analysis of these data revealed that the difference found between the two techniques was not significant, indicating that both can be used for quantification of ammonia-oxidizing bacteria. For nitrite-oxidizing bacteria it was not possible to make this comparison, since the bacterial genera that were being determined in each technique were likely different. Thus, MPN and FISH techniques proved to be relatively simple and suitable for quantification of nitrifying microorganisms in sludge samples, each of them with advantages and limitations.

An outbreak of hepatitis A virus among children in a flood rescue camp: A post‑disaster catastrophe.

We report an outbreak of acute viral hepatitis among children in a flood rescue camp at Rudraprayag district of Uttarakhand State, India. In May 2013, there was a disastrous natural calamity, The Himalayan Tsunami in Himalayan and Sub‑Himalayan region of Uttarakhand. More than 5700 people were feared dead, and thousands were sheltered in different rescue camps. A linkage was hypothesised between the infected individuals and the common water sources feared of being contaminated faecally. Aetiological agent of the present outbreak was HAV that is emerging in an outbreak form in India, emphasizing a definite need for formulating mandatory vaccination and proper control strategies. The report exemplifies the basic problems encountered after a natural calamity.

Número mais provável miniaturizado e microbiologia convencional para isolamento de Salmonella spp. em abatedouros de frangos de corte / Miniaturized most probable number and conventional microbiology for isolation of Salmonella spp. in poultry slaughterhouses

Os produtos de origem avícola podem ser importantes veículos de transmissão de Salmonella spp. para humanos e, dentre os vários parâmetros que determinam a qualidade de um alimento, destacam-se os que definem suas características microbiológicas. Objetivou-se detectar e quantificar Salmonella spp. na tecnologia de abate de frangos de corte por microbiologia convencional (MC) e número mais provável miniaturizado (mNMP). As coletas foram realizadas em duas visitas a três abatedouros sob Inspeção Federal e em seis pontos de coleta em triplicata, definidos como: recepção das aves (swabs de cloaca e esponjas de gaiolas de transporte antes e após a higienização) e carcaças (após pré resfriamento em chiller, após o gotejamento e antes da embalagem primária e congeladas a -12oC por 24 horas), totalizando 108 amostras...

Poultry products can be important modes of transmission of Salmonella spp. to humans and, among several parameters used to determine food quality, microbiological characteristics play an essential role. The aim of this study was to determine and quantify Salmonella spp. at broiler slaughtering facilities. This was done by conventional microbiology and by the miniaturized most probable number (mMPN) methods. Three federally-inspected slaughterhouses were visited, where samples were collected in triplicate from six sites: reception of live birds (cloacal swabs and sponge samples from transport cages before and after sanitation) and carcass processing (after pre-chiller, after dripping, and before primary packaging and refrigeration at -12oC for 24h), totaling 108 samples...

Prevalence and quantification of Listeria monocytogenes in beef offal at retail level in Selangor, Malaysia

A total of 63 beef offal samples (beef liver = 16; beef lung = 14; beef intestine = 9; beef tripe = 15; beef spleen = 9) from three wet markets (A, B, and C) in Selangor, Malaysia were examined for the prevalence and microbial load of Listeria monocytogenes. A combination of the most probable number and polymerase chain reaction (MPN-PCR) method was employed in this study. It was found that L. monocytogenes detected in 33.33% of the beef offal samples. The prevalence of L. monocytogenes in beef offal purchased from wet markets A, B, and C were 22.73%, 37.50% and 41.18% respectively. The density of L. monocytogenes in all the samples ranged from<3upto> 2,400 MPN/g. The findings in this study indicate that beef offal can be a potential vehicle of foodborne listeriosis.

Dinâmica de rizóbios em solo do cerrado de Roraima durante o período de estiagem / Dynamic of rhizobia in the soil during the dry season in cerrado of Roraima

The biological nitrogen fixation in legumes is performed by a group of bacteria known as rhizobia. The survival of these bacteria in soils is affected by several factors, such as temperature, drought and soil fertility. This study was performed to evaluate the dynamics of rhizobia in the soil after soybean cultivation and during a dry season in the cerrado of Roraima. Three areas were sampled: i) native cerrado as reference; ii) an area previously cultivated with soybean for one season; and iii) another one cultivated for two seasons also with soybean. The soil was sampled at a depth of 0-10 cm in five times (0, 45, 90, 135 and 180 days) during the dry season (September 2006 to March 2007). The rhizobial density in the soil was evaluated by the most probable number method with infection of soybean and cowpea plants. It was observed very low number of soybean nodulating bacteria in the reference area, but a high density, of up to several hundred rhizobia capable to nodulate cowpea was measured in this same area. Cropping of soybean with inoculated seeds increased rhizobial density evaluated by both trapping hosts. In cropped areas, an intense reduction of rhizobium density was observed just after soybean harvest, and this reduction continued until the end of the period of evaluation. It was concluded that soybean cultivation increases the density of rhizobial in the cerrado soil; however, this density is drastically reduced, during the dry season, by 99% at the end of the dry period.

A fixação biológica de nitrogênio que ocorre em leguminosas é realizada por um grupo de bactérias conhecidas como rizóbios. A sobrevivência destas bactérias no solo é influenciada por diversos fatores como a temperatura, umidade e fertilidade do solo. O objetivo deste trabalho foi avaliar a dinâmica da população de rizóbios em solo após o cultivo de soja, durante o período de estiagem no cerrado de Roraima. Foram amostradas três áreas: i) cerrado nativo como referência; ii) área cultivada uma vez com soja inoculada com rizóbio; iii) e área cultivada duas vezes com soja inoculada com rizóbio em anos consecutivos. O solo foi coletado na profundidade de 0-10 cm em cinco períodos a partir do inicio da estiagem no mês de setembro de 2006 coincidindo com a época de colheita da soja e prolongando-se até março de 2007 (0, 45, 90, 135 e 180 dias). A população de rizóbios no solo foi avaliada pela técnica do número mais provável (NMP) utilizando plantas de soja e de feijão-caupi como espécies isca. Foi observado que na área nativa praticamente não existiam bactérias nodulantes de soja, mas havia uma população capaz de nodular o feijão-caupi de até algumas centenas de rizóbios por gramas de solo. O cultivo da soja utilizando sementes inoculadas elevou a população de rizóbios no solo que foi constatada por ambas às espécies de plantas isca. Nas áreas cultivadas, constatou-se uma intensa redução da população de rizóbios no solo, em especial logo após a colheita da soja, continuando o decréscimo até o último período de avaliação. Conclui-se que o cultivo da soja inoculada com rizóbio eleva a densidade de rizóbios em solo do cerrado, mas durante a estiagem ocorre uma drástica redução dessa população, que pode chegar a mais de 99% considerando o início e final do período.

Calidad bacteriológica de aguas en piscinas públicas y privadas de la ciudad de Cumaná, estado Sucre, Venezuela / Bacteriological quality of water of public and private pools of the city of Cumaná, Sucre state, Venezuela

Para evaluar la calidad bacteriológica de aguas de piscinas en la ciudad de Cumaná, estado Sucre, Venezuela, se recolectaron muestras de agua en 1 piscina pública y 4 privadas, codificadas de la A a la E; se tomaron 2 muestras semanales durante 2 meses, antes y después de la limpieza. Se determinó pH, temperatura y cloro residual; los aerobios mesófilos por contaje en placas, el Número Más Probable (NMP) para coliformes totales (CT) y fecales (CF) e identificación bacteriana por métodos convencionales. El pH osciló entre 6,8 y 7,3, la temperatura de 29 a 31ºC y el cloro residual de 0,3 a 0,5 mg/L. El contaje más elevado de bacterias mesófilas se obtuvo en B con 6x10 2 UFC/mL, y el más bajo en C con 3x10 2 UFC/mL. En relación al NMP, antes de la limpieza, el valor más alto se obtuvo en D con 2,8x10 3 CT/100 ml; E mostró el valor más alto de CF /100 ml. Después de la limpieza B mostró el valor más alto de CT ubicándose en 9,3mLx10 2 y los valores más alto de CF para D y E en 3x10 2 . Los valores de CF antes y después de la limpieza superan lo establecido por la normativa Venezolana (0 NMP/100mL). Estadísticamente, no se evidenciaron diferencias significativas entre las piscinas para CT y CF antes y después de la limpieza. Las bacterias Gramnegativas predominaron, en E (84,21%) y B (71,92%), Klebsiella pneumoniae, Escherichia coli y Pseudomonas aeruginosa. A presentó mayor número de aislados Grampositivos (44,44%), identificándose Staphylococcus epidermidis y Enterococcus faecalis. Estos resultados indican una constante contaminación bacteriana y riesgo sanitario.

To evaluate the bacteriological quality of water pools in the city of Cumaná, Sucre state, Venezuela, water samples were collected in 1 public and 4 private pools, coded A to E, respectively, 2 samples were taken weekly for 2 months before and after a pool cleaning process. We determined pH, temperature and residual chlorine, aerobic mesophilic for total plate count, the Most Probable Number (MPN) for total coliforms (TC) and fecal (FC) and bacterial identification by conventional methods. The pH in the samples ranged between 6.8 and 7.3, temperature of 29 to 31ºC and chlorine residual of 0.3 to 0.5 mg/L The highest count of aerobic mesophilic bacteria was obtained in B with 6x10 2 . CFU/mL. In relation to the MPN, before cleaning, the CT highest value was obtained in D with 2.8 x10 3 and CT/100mL, E showed the highest CF value with 6x10 2 CF/100mL. After cleaning, the results for B indicated the CT highest values, 9.3 x10 2 CT/100mL, and in D and E the results had the CF highest values, 3x10 2 CF/100 mL. CF values in the samples before and after cleaning exceed the standards established by Venezuela (0 NMP/100mL). Statistically, no significant differences were found between pools for CT and CF before and after cleaning. Gram-negative bacteria predominated, being more frequent in E (84.21%) and B (71.92%), mainly, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. Sample A had more isolated Gram-positive (44.44%), identified as Staphylococcus epidermidis and Enterococcus faecalis. These results indicate a constant bacterial contamination and health risk.

Evaluation of microorganisms with sulfidogenic metabolic potential under anaerobic conditions

The aim of this work was to identify groups of microorganisms that are capable of degrading organic matter utilizing sulfate as an electron acceptor. The assay applied for this purpose consisted of running batch reactors and monitoring lactate consumption, sulfate reduction and sulfide production. A portion of the lactate added to the batch reactors was consumed, and the remainder was converted into acetic, propionic and butyric acid after 111 hours of operation These results indicate the presence of sulfate-reducing bacteria (SRB) catalyzing both complete and incomplete oxidation of organic substrates. The sulfate removal efficiency was 49.5% after 1335 hours of operation under an initial sulfate concentration of 1123 mg/L. The SRB concentrations determined by the most probable number (MPN) method were 9.0x10(7) cells/mL at the beginning of the assay and 8.0x10(5) cells/mL after 738 hours of operation.

Standardization of a quantification method for Salmonella spp. and Shigella spp. in specific liquid media / Estandarización de un método de recuento para Salmonella spp. y Shigella spp. en medios de cultivo líquidos especializados

Introduction: Chlorination is the most widely used disinfection process for drinking water production. The formation of chlorination carcinogenic by-products and chlorine intoxication by direct manipulation in small communities has motivated the study of alternative disinfection processes. In this sense, processes of advanced oxidation (PAOs) have yielded promising results. Escherichia coli (E. coli) is customarily used as faecal bacterial indicator to determine the efficiency of disinfection processes. However, it has been shown that E. coli is less resistant to disinfection than other enteric bacteria such as Shigella  spp. and Salmonella  spp. Additionally, the viable non-culturable (VNC) state yields bacteria which are not detectable on many culture media. Objective: The main objective is to standardize a method for counting Salmonella  spp. and Shigella  spp. in specific liquid media to reliably quantify the bacteriological potential risk related to disinfection processes based on PAO. Methods: The study followed a randomized bi-factorial experimental design and the Duncan multiple comparison test. This design allowed the selection of specific liquid media to fittingly standardize the counting of Salmonella  spp. and Shigella  spp. Results: We found that the best broth for counting Salmonella typhimurium strain at different concentrations in pure and mixed cultures was the Rappaport broth RP, the EE broth also allowed growing the two bacterial species tested in this research. Nonetheless, the latter results suggest the use of additional tests for this particular broth. Discussion: There was a variation in the counting results when pure cultures were used compared to those obtained from mixtures of microorganisms. It was also noted that Salmonella typhimurium and Shigella sonnei, were recovered from minimal concentrations in both RP and EE broths, respectively. To some extent, this suggests an additional confirmative method when using the EE® broth...

Introducción: La cloración es el método más usado para desinfectar aguas de consumo. La formación de subproductos cancerígenos y las intoxicaciones por manipulación directa en pequeñas comunidades, han motivado el estudio de procesos alternativos. Los procesos de oxidación avanzada (PAOS), han arrojado resultados prometedores, utilizando el indicador bacteriano Escherichia coli (E. coli), con el método recuento en placa. Sin embargo, también se ha demostrado que E. coli es menos resistente a la desinfección que otras bacterias entéricas como Shigella y Salmonella y que estos procesos generan bacterias viables que no se cultivan durante el proceso, y no se descubren en medios sólidos. Objetivo: Estandarizar un método de recuento de Salmonella sp. y Shigella sp., en medios de cultivo líquidos especializados, que permita valorar de forma confiable el riesgo bacteriológico en procesos de desinfección PAOS. Métodos: En el presente trabajo se ensayaron y seleccionaron medios líquidos especializados, con los que se estandarizó el recuento de Salmonella sp. y Shigella sp., mediante un diseño experimental aleatorizado bifactorial y la prueba de comparaciones múltiples de Duncan. Resultados: Se encontró que el mejor caldo para recuperar a S. typhimurium a diferentes concentraciones, en cultivos puros y mezclas, fue el caldo Rappaport de Merck (RP). El caldo de enriquecimiento para entero bacterias de Oxoid (EE), permitió un buen crecimiento de las dos especies objeto de esta investigación. Lo cual sugiere el empleo de pruebas adicionales cuando se use caldo EE para NMP. Discusión: Se observó una variación en el recuento cuando se usaron cultivos puros, comparado con la obtenida a partir de mezclas de microorganismos. Sin embargo, S. typhimurium. y Shigella sonnei logran ser recuperadas de concentraciones mínimas en los caldos RP, respectivamente...

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

Propagules of arbuscular mycorrhizal fungi in a secondary dry forest of Oaxaca, Mexico

Plant cover loss due to changes in land use promotes a decrease in spore diversity of arbuscular mycorrhizal fungi (AMF), viable mycelium and, therefore, in AMF colonization, this has an influence in community diversity and, as a consequence, in its recovery. To evaluate different AMF propagules, nine plots in a tropical dry forest with secondary vegetation were selected: 0, 1, 7, 10, 14, 18, 22, 25, and 27 years after abandonment in Nizanda, Oaxaca, Mexico. The secondary vegetation with different stages of development is a consequence of slash and burn agriculture, and posterior abandonment. Soil samples (six per plot) were collected and percentage of AMF field colonization, extrarradical mycelium, viable spore density, infectivity and most probable number (MPN) ofAMF propagules were quantified through a bioassay. Means for field colonization ranged between 40% and 70%, mean of total mycelium length was 15.7 +/- 1.88 mg(-1) dry soil, with significant differences between plots; however, more than 40% of extracted mycelium was not viable, between 60 and 456 spores in 100 g of dry soil were recorded, but more than 64% showed some kind of damage. Infectivity values fluctuated between 20% and 50%, while MPN showed a mean value of 85.42 +/- 44.17 propagules (100 g dry soil). We conclude that secondary communities generated by elimination of vegetation with agricultural purposes in a dry forest in Nizanda do not show elimination of propagules, probably as a consequence of the low input agriculture practices in this area, which may encourage natural regeneration.

La vegetación secundaria con diferentes grados de desarrollo es consecuencia de prácticas agrícolas de rozatumba- quema y su posterior abandono. La remoción de la vegetación por cambios de uso de suelo promueve una disminución en la diversidad de esporas, micelio viable y por lo tanto de la colonización de los hongos micorrizógenos arbusculares (HMA), lo cual repercute en la diversidad de la comunidad y como consecuencia en su regeneración. Para evaluar los propágulos de HMA se seleccionaron nueve parcelas con vegetación secundaria con diferentes edades de abandono: 0, 1, 7, 10, 14, 18, 22, 25, 27 años, en la región de Nizanda, Oaxaca, México. Se recolectaron muestras de suelo (seis por parcela) y se cuantificó la colonización de campo, el micelio extrarradical, la densidad de esporas viables, así como la infectividad y el número más probable de propágulos infectivos (NMP). Los promedios de la colonización de campo fueron de 40 a 70 %, el promedio de la longitud de micelio total alcanzó 15.7 ± 1.88 mg-1 suelo seco con diferencias significativas entre parcelas, pero más del 40 % del micelio extraído no fue viable. Se encontraron entre 60 y 456 esporas en 100 g de suelo pero más del 64 % presentaron algún tipo de daño. Los valores de infectividad se encontraron entre 20 % y 50 %, mientras que el NMP presentó un promedio de 85.42 ±44.17 (100 g de suelo seco). Concluimos que las comunidades secundarias generadas por la eliminación de la vegetación con fines agrícolas en la selva baja caducifolia en Nizanda o presentan eliminación de propágulos, probablemente por el bajo impacto de la agricultura, lo cual indica que la regeneración natural es posible.

Comparación de los métodos PetrifilmTM coliformes y Número Más Probable (NMP) para la determinación de coliformes fecales en muestras de queso blanco / Comparison study between the coliform PetrifilmTM and the Most Probable Number methods for the assessment of fecal coliforms in white cheese

Los coliformes fecales son un grupo importante de microorganismos indicadores de inocuidad en alimentos, constituido principalmente por Escherichia coli, el cual es considerado como indicador de contaminación reciente de origen fecal, por ello la importancia de investigar su presencia y determinar rápidamente el nivel poblacional en alimentos. Este trabajo tiene como objetivo comparar el método tradicional Número Más Probable (NMP) para la determinación de coliformes fecales, según Norma Venezolana Covenin Nº 1104-96 con el método de placas rehidratables PetrifilmTM 3M coliformes incubadas en baño de agua circulante (PBA) a 45 ± 0,2°C por 24 ± 2 horas y en estufa con aire circulante 44 ± 1°C por 24 ± 2 horas (PES) de acuerdo con lo recomendado por la Asociación Francesa de Normalización (AFNOR) y la Corporación 3M. Se analizaron un total de 42 muestras de queso blanco aplicando ambas metodologías, dispensando simultáneamente diluciones seriadas de las muestras. Para el análisis estadístico se realizó una correlación de muestras relacionadas (SPS versión 7,5), obteniéndose los siguientes resultados r = 0,952 entre NMP y PES y r = 0,944 entre NMP y PBA; lo que indica una buena correlación positiva entre ambas metodologías en sus diferentes modalidades para la determinación de coliformes fecales en muestras de queso blanco. Se concluye que las placas PetrifilmTM coliformes incubadas a la temperatura óptima de crecimiento de dichos microorganismos es un método alternativo, rápido y confiable para la determinación del nivel de coliformes fecales en queso blanco.

Fecal coliform belong to an important group of sanitary quality indicator microorganisms in food, mainly constituted for Escherichia coli, considerated as indicators of recent fecal contamination, that is why it is very important to investigate their presence and to detect their population in food rapidly. The objetive of this study was to compare the Most Probable Number (NPM) method for fecal coliform determination, according to Venezuelan standard COVENIN Nº 1104-96, with the coliform PetrifilmTM 3M TM plates, incubated in circulating thermostatically - controlled water bath (PBA) at 45 ± 0,2 ° C for 24 ± 2 hours and in a circulating air incubator at 44 ± 1° C for 24 ± 2 hours (PES), according to the recommendation of Association Francoise of Normalization, Paris (AFNOR) and 3M TM corporation. Forty-two white cheese samples were analyzed using both methods mentioned above. They were dispensed at the same time with decimal dilutions of the samples. Data generated were subjected to correlation of related samples (SPS 7.5 version) and the correlation coefficients (r) were obtained; r = 0.952 NMP and PES; r = 0.944 NMP and PBA. It is interesting to observe a good correlation between the methodologies in their different forms for fecal coliform determination in white cheese. Coliform PetrifilmTM plates incubated at the optimal temperature of coliform fecal culture represent a rapid alternative and reliable method for the assessment of fecal coliform population in white cheese.