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.

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.

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.

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.

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.