Persistence of Fecal Contamination Indicators and Pathogens in Class B Biosolids Applied to Sugarcane Fields.

Agricultural recycling of human Class B biosolids in sugarcane ( spp.) crop is a promising alternative to reduce the costs of biosolids disposal. However, the presence of fecal contamination indicators such as thermotolerant coliforms and pathogenic organisms such as enterovirus and spp. in biosolids impose barriers to effective and widespread use of biosolids as fertilizer. In addition, there is a scarcity of studies that investigate the persistence of these organisms in tropical soils. This study aimed to evaluate the persistence of pathogenic and fecal indicators for 258 d in a tropical clayey soil amended with human Class B biosolids and cultivated with sugarcane. Treatments were immediate incorporation of biosolids into soil after application (T1) or superficial application of biosolids followed by incorporation after 35 d (T2), emulating the typical procedure in sugarcane fields. Thermotolerant coliforms were estimated to persist for 437 d in T1 and 398 d in T2. For enterovirus, mean estimated persistence time in soil was 26 d for T1, but the sampling frequency was insufficient in T2 for persistence analysis. After 35 d, no enterovirus was detected in any sample. Mean estimated persistence time for viable spp. eggs in soil was 22 d in T1 and 41 d in T2.

Persistence of Ascaris spp. Ova in Tropical Soil Cultivated with Eucalyptus and Fertilized with Municipal Biosolids.

In many countries, the main reason for severely restricting or outright banning the land application of class B biosolids is the lack of risk assessment for adverse human health impacts. Among pathogens that are not often studied are helminth ova, including that of the spp. Almost all of the knowledge about the persistence of spp. ova in soils fertilized with biosolids is based on studies developed in North America, Europe, and Asia. These studies have almost always been conducted under temperate climate conditions, which may cause erroneous interpretations when the conclusions are extrapolated to tropical regions such as those found in Brazil. This team evaluated the persistence of viable spp. ova in a sandy Quartzipsamment tropical soil, previously planted with × hybrid () and fertilized with biosolids, over a 52-wk period. During the reporting period, the average temperature of soil and biosolids fluctuated between 15 and 30°C, and the average moisture of biosolids fluctuated between 60 and 90%. The estimated persistence time of viable spp. ova after land application was estimated at close to 7 wk, indicating that ova may not be viable for as long as it has been shown to be in studies of more temperate areas. The relationship of temperature with persistence of viable spp. ova in a tropical soil was stronger than moisture content, suggesting that temperature substantially contributed to their nonviability over the course of the experiment.

Characterization of Enterococcus species isolated from marine recreational waters by MALDI-TOF MS and Rapid ID API® 20 Strep system.

MALDI-TOF Mass Spectrometry Biotyping has proven to be a reliable method for identifying bacteria at the species level based on the analysis of the ribosomal proteins mass fingerprint. We evaluate the usefulness of this method to identify Enterococcus species isolated from marine recreational water at Brazilian beaches. A total of 127 Enterococcus spp. isolates were identified to species level by bioMérieux's API® 20 Strep and MALDI-TOF systems. The biochemical test identified 117/127 isolates (92%), whereas MALDI identified 100% of the isolates, with an agreement of 63% between the methods. The 16S rRNA gene sequencing of isolates with discrepant results showed that MALDI-TOF and API® correctly identified 74% and 11% of these isolates, respectively. This discrepancy probably relies on the bias of the API® has to identify clinical isolates. MALDI-TOF proved to be a feasible approach for identifying Enterococcus from environmental matrices increasing the rapidness and accuracy of results.

Occurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru.

The objectives of the study were to detect and genotype Cryptosporidium spp. and Giardia intestinalis in wastewater samples obtained from five cities with high transit of people in the State of São Paulo, Brazil, and at the entrance of a Wastewater Treatment Plant (WWTP) in Lima, Peru. Samples were collected and concentrated by centrifugation. The genomic DNA was extracted for molecular characterization by nested PCR for Cryptosporidium and double nested PCR for Giardia, followed by sequencing and phylogenetic analysis. G. intestinalis was found in 63.6 % of the samples, and the human assemblages A and B were identified. Cryptosporidium sp. was found in 36.4 % of the samples, and the species were corresponding to Cryptosporidium hominis, Cryptosporidium cuniculus, and Cryptosporidium muris. Results revealed the presence of human pathogenic Cryptosporidium species and G. intestinalis human pathogenic assemblages. Molecular tools highlight the importance to map the genetic diversity of these parasites, as well as to detect their epidemiological circulation pathway in the environment.

Molecular and Phenotypic Characterization of a Highly Evolved Type 2 Vaccine-Derived Poliovirus Isolated from Seawater in Brazil, 2014.

A type 2 vaccine-derived poliovirus (VDPV), differing from the Sabin 2 strain at 8.6% (78/903) of VP1 nucleotide positions, was isolated from seawater collected from a seaport in São Paulo State, Brazil. The P1/capsid region is related to the Sabin 2 strain, but sequences within the 5'-untranslated region and downstream of the P1 region were derived from recombination with other members of Human Enterovirus Species C (HEV-C). The two known attenuating mutations had reverted to wild-type (A481G in the 5'-UTR and Ile143Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype and had accumulated amino acid substitutions in neutralizing antigenic (NAg) sites 3a and 3b. The date of the initiating OPV dose, estimated from the number of synonymous substitutions in the capsid region, was approximately 8.5 years before seawater sampling, a finding consistent with a long time of virus replication and possible transmission among several individuals. Although no closely related type 2 VDPVs were detected in Brazil or elsewhere, this VDPV was found in an area with a mobile population, where conditions may favor both viral infection and spread. Environmental surveillance serves as an important tool for sensitive and early detection of circulating poliovirus in the final stages of global polio eradication.

Clustering of water bodies in unpolluted and polluted environments based on Escherichia coli phylogroup abundance using a simple interaction database.

Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A1 and B23 in rivers impacted by human pollution sources, while subgroups D1 and D2 were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.

Draft Genome Sequence of Non-O1 and Non-O139 Vibrio cholerae Strain VCC19.

Vibrio cholerae O1 is the causative agent of cholera and is ubiquitous in the aquatic environment, while V. cholerae strains non-O1 and non-O139 are recognized as causative agents of sporadic and localized outbreaks of diarrhea. Here, we report the complete sequence of a non-O1 and non-O139 V. cholerae strain (VCC19), which was isolated from the environment in Brazil. The sequence includes the integrative conjugative element (ICE). This paper is the first report of the presence of such an element in a V. cholerae strain isolated in Brazil.

Pathogenic parasites and enteroviruses in wastewater: support for a regulation on water reuse.

Brazilian regulations for nonpotable reuse are being established using World Health Organization guidelines, however, they should be developed based on local monitoring studies. This study intended to analyze enteroviruses, protozoa and viable Ascaris sp. eggs in raw (24) and treated (24) effluents from four Wastewater Treatment Plants of São Paulo State, Brazil. The protozoa were detected with the US Environmental Protection Agency (USEPA) Method 1623 in the treated effluents and by centrifugation/Immunomagnetic Separation in the raw influent samples. Viable Ascaris sp. eggs were analyzed according to a modified USEPA method. Enteroviruses were quantified by using human rhabdomyosarcoma cells after adequate concentration procedures. All wastewater influents were positive for Giardia sp. whereas Cryptosporidium sp. was detected in 58.3% of the samples. Giardia sp. and Cryptosporidium sp. were present in 79.2 and 25.0% respectively, of the treated wastewater samples. Viable Ascaris sp. eggs were detected in 50.0 and 12.5% of influent and treated wastewater samples. Enteroviruses were isolated in the 24 raw influent samples and in 46% of the treated samples. Taking into account the densities of Giardia sp. in some treated wastewaters intended to be used as reclaimed water, Quantitative Microbial Risk Assessment studies should be conducted to establish pathogen quantitative criteria for a future Brazilian regulation for water reuse.

Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil.

A survey of Giardia and Cryptosporidium was conducted in surface water used as drinking water sources by public water systems in four densely urbanized regions of Sao Paulo State, Brazil. A Quantitative Microbial Risk Assessment, based on protozoa concentrations, was performed to estimate the probability of protozoa infection associated with drinking water ingestion. A total of 206 source water samples were analyzed over a 24 month period using the USEPA Method 1623. The risk of infection was estimated using an exponential dose response model, children and adults exposure and a gamma distribution for (oo)cyst concentrations with three scenarios for treating censored data. Giardia was detected in 102 of the samples, and 19 of them were also positive for Cryptosporidium, with maximum concentrations of 97.0 cysts/L and 6.0 oocysts/L, respectively. Risk distributions were similar for the three scenarios. In the four regions, the estimated risk of Giardia infection per year, for adults and children, ranged from 0.29% to 2.47% and from 0.08% to 0.70%, respectively. Cryptosporidium risk infection varied from 0.15% to 0.29% for adults and from 0.04% to 0.08% for children. In both cases, the calculated risk surpassed the risk of infection of 10(-4) (1:10,000) defined as tolerable by USEPA for a yearly exposure. The probability of Giardia infection was very close to the rates of acute diarrheic disease for adults (1% to 3%) but lower for children (2% to 7%). The daily consumption of drinking water was an important contributing factor for these differences. The Microbiological Risk Assessment carried out in this study provides an indication of infection risks by Giardia and Cryptosporidium in the population served by these source waters. Strategies for source water protection and performance targets for the water treatment should be established to achieve the required level of public health risk.

Avaliação da performance de metodologias de detecção de Cryptosporidium spp. e Giardia spp. em água destinada ao consumo humano, para o atendimento às demandas da Vigilância em Saúde Ambiental no Brasil / Performance evaluation of different methodologies for detection of Cryptosporidium spp. and Giardia spp. in water for human consumption to meet the demands of the Environmental Health Surveillance in Brazil

Objetivo: avaliar a performance de três protocolos de concentração de cistos e oocistos em amostras de água bruta de rios brasileiros. Métodos: os protocolos estudados foram precipitação química, filtração em membranas de 47mm de diâmetro e 3μm de porosidade nominal e filtração com o sistema Filta-Max®. Amostras de água bruta coletadas de rios nos estados de São Paulo e Minas Gerais foram analisadas após contaminação artificial. Os resultados de precisão inicial e recuperação de organismos marcados (Color-Seed®) foram comparados com os critérios da Agencia de Proteção Ambiental dos Estados Unidos da Améri¬ca (USEPA). Resultados: Nos ensaios de precisão inicial para cistos de Giardia, a filtração em membranas e com Filta-Max® atingiram os critérios da USEPA para recuperação de cistos. Para Cryptosporidium, somente o procedimento com Filta-Max® alcançou os critérios. Conclusão: o sistema Filta-Max® foi o único método que atingiu todos os critérios para identificação de Cryptosporidium e Giardia em água.

Objective: this study aimed to evaluate three different concentration protocols of cysts and oocysts in raw water samples in Brazilian rivers. Methods: the protocols studied were chemical precipitation, filtration in membranes of 47mm of diameter versus nominal porosity of 3µm and filtration using Filta-Max® system. The raw water samples collectedfrom rivers in the states of São Paulo andMinas Gerais were analyzed after being seeded with Color-Seed®. The results of initial precision using filtration in membranes and recovery of Color-Seed® organisms were compared to the acceptance criteria established by the United States Environmental Protection Agency (USEPA). Results: The study of initial precision to Giardia using filtration in membranes and recovery of Color-Seed® met the USEPA criteria; for Cryptosporidium only the Filta-Max® achieved the established criteria. Conclusion: Filta-Max® system showed to be the only method that achieved all the performance criteria for identification of Giardia and Cryptosporidium in water.

Comparison of thermotolerant coliforms and Escherichia coli densities in freshwater bodies

Fecal bacterial indicator analyses have been widely used for monitoring the water quality. This study was designed to determine the ratio between the density of Escherichia coli and other Thermotolerant Coliforms (TtC) bacteria from freshwater samples collected for a two-year period of monitoring. TtC were enumerated by membrane filtration on mFC agar. E. coli enumeration was done by two methods: TtC colonies identified in mFC were inoculated in EC-MUG or water samples were filtered and inoculated in modified mTEC agar media, and both methods were compared for quantitative recovery of E. coli. The results pointed out a mean percentage of E. coli among other thermotolerant coliforms (E. coli/TtC ratio) of 84.3% in mFC media. Taking these results into account, a mandatory standard of 1000 thermotolerant coliforms would correspond to 800 E. coli and the adoption of these E. coli based standards will represent a major improvement for the monitoring of freshwater quality.

Comparison of thermotolerant coliforms and Escherichia coli densities in freshwater bodies.

Fecal bacterial indicator analyses have been widely used for monitoring the water quality. This study was designed to determine the ratio between the density of Escherichia coli and other Thermotolerant Coliforms (TtC) bacteria from freshwater samples collected for a two-year period of monitoring. TtC were enumerated by membrane filtration on mFC agar. E. coli enumeration was done by two methods: TtC colonies identified in mFC were inoculated in EC-MUG or water samples were filtered and inoculated in modified mTEC agar media, and both methods were compared for quantitative recovery of E. coli. The results pointed out a mean percentage of E. coli among other thermotolerant coliforms (E. coli/TtC ratio) of 84.3% in mFC media. Taking these results into account, a mandatory standard of 1000 thermotolerant coliforms would correspond to 800 E. coli and the adoption of these E. coli based standards will represent a major improvement for the monitoring of freshwater quality.

Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo state, Brazil.

Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, Al and All. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype All from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype All was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.

Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination.

BACKGROUND: Escherichia coli strains are commonly found in the gut microflora of warm-blooded animals. These strains can be assigned to one of the four main phylogenetic groups, A, B1, B2 and D, which can be divided into seven subgroups (A0, A1, B1, B22, B23, D1 and D2), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Distinct studies have demonstrated that these phylo-groups differ in the presence of virulence factors, ecological niches and life-history. Therefore, the aim of this work was to analyze the distribution of these E. coli phylo-groups in 94 human strains, 13 chicken strains, 50 cow strains, 16 goat strains, 39 pig strains and 29 sheep strains and to verify the potential of this analysis to investigate the source of fecal contamination. RESULTS: The results indicated that the distribution of phylogenetic groups, subgroups and genetic markers is non-random in the hosts analyzed. Strains from group B1 were present in all hosts analyzed but were more prevalent in cow, goat and sheep samples. Subgroup B23 was only found in human samples. The diversity and the similarity indexes have indicated a similarity between the E. coli population structure of human and pig samples and among cow, goat and sheep samples. Correspondence analysis using contingence tables of subgroups, groups and genetic markers frequencies allowed the visualization of the differences among animal samples and the identification of the animal source of an external validation set. The classifier tools Binary logistic regression and Partial least square--discriminant analysis, using the genetic markers profile of the strains, differentiated the herbivorous from the omnivorous strains, with an average error rate of 17%. CONCLUSIONS: This is the first work, as far as we are aware, that identifies the major source of fecal contamination of a pool of strains instead of a unique strain. We concluded that the analysis of the E. coli population structure can be useful as a supplementary bacterial source tracking tool.

Aeromonas spp. and microbial indicators in raw drinking water source

Aeromonas species are autochtonous in the aquatic environment and some of them have been associated with health effects like wound infections, septicemia and diarrhoeal illness. In this study, the occurrence of Aeromonas spp. and microbial indicators in raw drinking water from wells, springs, fountains and mineral waters was evaluated. A total of 126 water samples was analyzed for Aeromonas spp. by the membrane filtration technique using ADA media and by P/A test. Typical colonies of Aeromonas spp. were submitted to biochemical tests for species differentiation. Toxin production was tested using Y-1 mouse adrenal cells. Coliforms and heterotrophic bacteria were enumerated by membrane filtration and pour plate techniques, respectively. P. aeruginosa, C. perfringens and fecal streptococci were determined by P/A method. Aeromonas spp. were isolated in 36.5 percent of the samples, whereas total and thermotolerant coliforms were detected in 51.2 percent and in 23.8 percent of the samples, respectively. C. perfringens, fecal streptococci and P. aeruginosa were present in 16.5 percent, 20.4 percent and 3.8 percent of the samples, respectively. The concentrations of heterotrophic bacteria were higher than 1,0x10³ CFU/mL in 52.5 percent of the samples. A. hydrophila was the most frequent species, followed by A. allosaccharophila,A. jandaei,A.sobria and HG2. A heat label toxin was detected in 13 from the 58 strains tested. These data show that the drinking water sources analyzed can represent a risk for human health. It is important to consider that wells and springs are used as drinking water supply in poor areas and rural regions, where undernourished people more susceptible to infections by these microorganisms predominate.

Bactérias do gênero Aeromonas são naturais no ambiente aquático e algumas espécies podem causar infecções em humanos como feridas, septicemia e diarréia. Este trabalho objetivou avaliar a ocorrência de Aeromonas sp. em 126 amostras de água de poços, nascentes, fontes e água mineral, e associar sua presença com indicadores microbianos de contaminação. Foi utilizada a técnica de membrana filtrante com o meio ADA e o teste P/A. Colônias típicas de Aeromonas sp. foram submetidas a testes bioquímicos para identificação da espécie. A produção de toxina foi avaliada utilizando-se células Y-1 de adrenal de camundongo. Coliformes e bactérias heterotróficas foram analisados através de filtração em membrana e pela técnica de inoculação em profundidade, respectivamente. P. aeruginosa, C. pefringens e os estreptococos fecais foram determinados pelo teste P/A. Aeromonas sp. foi isolada em 36,5 por cento das amostras, enquanto que os coliformes totais e termotolerantes estavam presentes em 51,2 por cento e 23,8 por cento das amostras, respectivamente. C. perfringens, estreptococos fecais e P. aeruginosa foram detectados em 16,5 por cento, 20,4 por cento e 3,8 por cento das amostras respectivamente. Concentrações de bactérias heterotróficas superiores a 1,0x10³ UFC/mL ocorreram em 52,5 por cento das amostras. A. hydrophila foi a espécie mais isolada, seguida por A. allosaccharophila, A. jandaei, A. sobria e HG2. Uma toxina termolábil foi detectada em 13 dos 58 isolados analisados. Portanto, as fontes de água de consumo humano analisadas podem representar um risco para a saúde humana. É importante considerar que fontes, poços e nascentes são utilizadas como suprimento de água em áreas pobres e regiões rurais, onde predominam pessoas com problemas de desnutrição, mais suscetíveis a doenças infecciosas.

Sanitary quality of sands from marine recreational beaches of São Paulo, Brazil

A sanitary evaluation of sand and water from 16 beaches of São Paulo State, Brazil, was undertaken during spring of 1997 and summer of 1998. Ninety six samples each of wet and dry sand and seawater were collected and analysed for fecal indicator bacteria. A parasitological examination and Candida albicans analysis were also performed in sand samples and F-specific bacteriophages were determined in seawater. Statistical analysis of the results demonstrated higher concentrations of fecal coliforms and fecal streptococci in dry sand during summer. Correlation analysis indicated a significant relationship between fecal indicator densities in wet sand and seawater. There was a significant correlation between the densities of fecal coliforms and fecal streptococci for both types of sand, and this correlation was higher in wet sand. Cysts and eggs of parasites were detected in 4.2 percent of the samples and Candida albicans was isolated in 18 percent of the samples. The high concentrations of fecal indicators detected in sand during summer demonstrate that there is a health risk to the users of these recreational areas and suggest the necessity of some criteria for microbiological control. Preventive measures, such as education campaings and some management actions are important precautionary measures.

Avaliaçäo da presença dos protozoários Giardia sp e Cryptosporidium sp em águas superficiais destinadas a captaçäo e tratamento para consumo humano no Estado de Säo Paulo / Evaluation of Giardia sp and Cryptosporidium sp in source water from Säo Paulo State, Brazil

Objetivo. Avaliar a presença dos protozoários Giardia sp e Cryotosporidium sp em águas destinadas a captaçäo e tratamento para consumo humano no Estado de Säo Paulo, como parte do projeto da CETESB (Companhia de Tecnologia e Saneamento Básico do Estado de Säo Paulo) "Avaliaçäo da Qualidade das Aguas Interiores do Estado de Säo Paulo". Trata-se de importantes agentes etiológicos de gastroenterites de veiculaçäo hídrica, amplamente disseminados no ambiente e com características que favorecem a transmissäo pela água. Métodos. Durante 19 meses foram analisadas 278 amostras de águas superficiais provenientes de 28 pontos de captaçäo de 10 bacias hidrográficas. Foi utilizada a técnica de imonofluorescência, após concentraçäo das amostras por floculaçäo por carbonato de cálcio. Foram igualmente , analisados para verificar-se eventual correlaçäo com esses protozoários, coliformes fecais, estreptococos fecais e Clostridium perfrigens. Resultados. Os resultados obtidos indicaram a presença da Giardia sp e Cryptosporidium sp, respectivamente em 27 por cento e 2,5 por cento das amostras analisadas, uma porcentagem de positividade inferior àquela relatada na literatura, principalmente para o Cryptosporidium sp. O teste de postos de Spearman revelou uma correlaçäo significativa, entre as concentraçöes de Giardia sp e os três indicadores de contaminaçäo fecal. Conclusöes. Segundo a Legislaçäo Americana de Monitoramento - ICR ( Information Collection Rule), em 16 dos 28 pontos de captaçäo avaliados seria necessário realizar análise da água tratada para avaliar a eficiência do tratamento na remoçäo dos parasitas. A complexidade dos métodos de analise requer rigoroso programa de Controle de Qualidade Analítica. Algumas deficiências técnicas do método ainda limitam a utilizaçäo os resultados do monitoramento em Saúde Pública.