Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157.

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters.

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

A multi-tiered approach to assess fecal pollution in an urban watershed: Bacterial and viral indicators and sediment microbial communities.

Development of effective pollution mitigation strategies require an understanding of the pollution sources and factors influencing fecal pollution loading. Fecal contamination of Turkey Creek in Gulfport, Mississippi, one of the nation's most endangered creeks, was studied through a multi-tiered approach. Over a period of approximately two years, four stations across the watershed were analyzed for nutrients, enumeration of E. coli, male-specific coliphages and bioinformatic analysis of sediment microbial communities. The results demonstrated that two stations, one adjacent to a lift station and one just upstream from the wastewater-treatment plant, were the most impacted. The station adjacent to land containing a few livestock was the least impaired. While genotyping of male-specific coliphage viruses generally revealed a mixed viral signature (human and other animals), fecal contamination at the station near the wastewater treatment plant exhibited predominant impact by municipal sewage. Fecal indicator loadings were positively associated with antecedent rainfall for three of four stations. No associations were noted between fecal indicator loadings and any of the nutrients. Taxonomic signatures of creek sediment were unique to each sample station, but the sediment microbial community did overlap somewhat following major rain events. No presence of Escherichia coli (E. coli) or enterococci were found in the sediment. At some of the stations it was evident that rainfall was not always the primary driver of fecal transport. Repeated monitoring and analysis of a variety of parameters presented in this study determined that point and non-point sources of fecal pollution varied spatially in association with treated and/or untreated sewage.

Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

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.

Transferencia de genes plasmídicos de resistencia a antibióticos mediante conjugación entre cepas de Escherichia coli provenientes de procesos diarréicosde niños menores a 3 años de edad. Hospital Materno Infantil de la ciudad de La Paz / Transfer of plasmid genes of resistance to antibiotics through conjugation between strains of escherichia coli from processes diarréicos de smaller children than 3 years of edad. Hospital Maternal and Child of the city of La Paz

Escherichia coli es el agente etiológico enterobacteriano más común causante de enfermedades diarreicas en lapoblación infantil. Por este motivo se estudió la transferencia por conjugación de genes plasmídicos de resistencia a antibióticos de bacterias E. coli provenientes de heces de niños menores de 3 años con procesos diarreicos que asistieron al laboratorio clínico delHospital materno infantil durante abril y diciembre del 2003. Se analizó el antibiograma de 44 cepas de E. colique presentó altos porcentajes de resistencia (Amoxicilina 95.55%, cotrimoxazol 68.88%, cloranfenicol51.11%). Por otra parte, las asociaciones más comunes de antibióticos fueron amoxicilina-cotrimoxazol (65%), y amoxicilina-cotrimoxazol-cloranfenicol (47,73%). Los experimentos de conjugación presentaron frecuencias entre 10-5 a 1 0-7, siendo de mayor valor para latransferencia de amoxicilina y menor para la amikacina. Los porcentajes de resistencia y las frecuencias deconjugación demuestran que la mayoría de los genes de resistencia son capaces de ser transferidos por conjugación. Lo que implica probablemente que muchosde estos genes están localizados en elementos genéticos como plásmidos y transposones.

Avaliaçäo da qualidade higiênico-sanitária da água de poços rasos localizados em uma área urbana: utilizaçäo de colifagos em comparaçäo com indicadores bacterianos de poluiçäo fecal / The evaluation of the hygienic and sanitary quality of water from private shallow wells situated in an urban area of south-eastern Brazil: a comparison between the use of coliphages and bacterian indicators of fecal pollution

Foram submetidas às contagens de colifagos, coliformes totais, coliformes fecais, e de estreptococos fecais, 104 amostras de água colhidas de 8 poços rasos localizados na área urbana do Município de Jaboticabal, SP, Brasil, com a finalidade de avaliar as condiçöes higiênico-sanitárias e de verificar as correlaçöes existentes entre o número de colifagos e o de bactérias indicadoras de poluiçäo fecal. Os resultados obtidos evidenciaram a ocorrência de 96 (92,3 por cento) amostras fora dos padröes bacteriológicos de potabilidade estabelecidos pelo Ministério da Saúde, mostrando ser precárias as condiçöes higiênico-sanitárias das aguás analisadas. Os achados evidenciaram a inexistência de correlaçäo entre o número de colifagos e os números de bactérias indicadoras de poluiçäo fecal

Qualidade bacteriológica de águas subterrâneas em cemitérios / Bacteriological quality of groundwater in cemiteries

Foram analisadas amostras de águas subterrâneas de três cemitérios localizados em áreas geologicamente distintas de Säo Paulo e de Santos, Brasil, com relaçäo às condiçöes higiênicas e sanitárias. Para as primeiras foram considerados os coliformes totais, bactérias heterotróficas, microrganismos proteolíticos e lipolíticos. Para as sanitárias foram pesquisados coliformes fecais, estreptococos fecais, clostrídios sulfito redutores, colifagos e salmonelas. Verificou-se que as águas näo apresentaram condiçöes higiênicas satisfatórias e, em alguns casos, foram encontrados níveis altos de nitrato (75,7 mg/l). A detecçäo de níveis mais elevados de estreptococos fecais e de clostrídios sulfito redutores em relaçäo aos coliformes fecais, na maior parte das amostras, parece mostrar que os dois primeiros indicadores seriam mais adequados para avaliaçäo das condiçöes sanitárias deste tipo de água. Foi detectada Salmonella apenas em uma amostra e näo foram detectados colifagos. Na análise estatística, foram encontradas correlaçöes significantes entre três indicadores de poluiçäo fecal assim como entre as contagens em placas de bactérias heterotróficas aeróbias, anaeróbias e lipolíticas. Foi observada uma relaçäo direta entre a deterioraçäo da qualidade da água e as condiçöes geológicas e hidrogeológicas do ambiente estudado, devendo este fator ser considerado para o planejamento e implantaçäo de cemitérios

Fotocatálise heterogênea com TiO2 aplicada ao tratamento de esgoto sanitário secundário / TiO2 heterogeneous photocatalysis in secondary wastewater treatment

Neste trabalho são apresentados resultados da aplicação da fotocatálise heterogênea utilizando dióxido de titânio imobilizado sobre placa de vidro, irradiado por luz solar, para desinfecção de esgoto sanitário secundário. Utilizou-se o efluente de um reator anaeróbio/aeróbio com leito expandido de carvão ativado que trata o esgoto do Campus da USP/São Carlos, e também de contribuições domésticas. Os experimentos foram do tipo batelada, com tempo de recirculação de 4 horas. As vazões estudadas foram 15, 22,5 e 30 L/h. Foram avaliadas as remoções de matéria orgânica (DQO e COT) e a inativação de microrganismos indicadores de contaminação fecal (coliformes totais, E. coli, Clostridium perfringens e colifagos).

Utilización de coliformes fecales y colifagos en el control de la calidad bacteriológica de aguas servidas tratadas por lodos activados / Use of fecal coliforms and coliphage to determine the bacteriological quality of activated sludge sewage treatment

Evidence from the scientific literature suggests that coliform bacteria are not adequate indicators for fecal contamination, and the need has been expressed for development of rapid, accurate, and cost-effective techniques for determining the bacteriological quality of water. One alternative has been the use of coliphage methods. The objetive of the present study was to evaluate bacteriological water quality of sewage treated with fecal coliforms and coliphage. Water samples were collected from three stations: 1) influent, 2) middle, and 3) effluent at the Antofagasta activated sludge treatment plant. Coliform bacteria were determined using the MPN technique, and coliphage enumeration as described by Isbister et al., (1983). Escherichia coli ATCC 13706 was used as the host strain. The counts of both organisms decreased in a similar way through the different treatment steps with little variation over a one-year period. Coliphage numbers were lower than the bacteria by about one log. After the sewage treatment chlorination step the coliphage number was higher than the fecal coliforms. These differences showed that the coliphage was more resistent to chlorine than the fecal coliforms. The results suggested than the coliphage could be used as an alternate indicator in evaluation of bacteriological quality of treated sewage effluent.

Fuertes evidencias sugieren que las bacterias coliformes no son indicadores adecuados de contaminación fecal. Hay una gran necesidad por el desarrollo de una técnica más rápida, acuciosa y de bajo costo para determinar la calidad bacteriológica del agua. Los colifagos parecen ser una excelente alternativa. El objetivo de este estudio fue evaluar la calidad bacteriológica de aguas servidas tratadas por medio de los coliformes fecales y colifagos. Muestras de aguas fueron recolectadas de tres estaciones: afluente, en el medio y efluente en la planta de tratamientos de Antofagasta por lodos activados. Las bacterias coliformes fueron determinadas por la técnica del NMP y la enumeración de colifagos, como describe Isbister y col (1983). Escherichia coli ATCC 13706 fue usada como cepa huésped. Los recuentos de ambos microorganismos disminuyeron en forma similar a lo largo de las diferentes etapas del tratamiento con variaciones pequeñas durante un año, siendo el número de colifagos más bajos que los coliformes fecales en un log. Al final del tratamiento de las aguas servidas, etapa de cloración, los números de los colifagos fueron más altos que los coliformes fecales. Estos resultados sugieren que los colifagos pueden ser usados como un indicador alternativo para la evaluación de la calidad bacteriológica de las aguas tratadas.