Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria.
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Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems. (+info)
Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.
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Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems. (+info)
The use of urban sewage sludge on pastures: the cysticercosis threat.
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Urban sewage production is increasing and its agronomical use as a fertiliser has been advocated. Considerable defiance is prevalent in consumers and among farmers on the use of such fertilisers due to unknown pathological or environmental risks. The aim of the present review was to consider which pathological risk is major. Cysticercosis due to Taenia saginata appears to be one of the major pathological threats when sewage sludge is used to fertilise cattle pastures in temperate areas. The situation is different in Africa (Taenia solium and T. saginata are both highly prevalent) and Asia (Taeniasaginata-like are prevalent). The processing of sludge and the delay between its application onto a pasture and grazing are probably major risk factors. Little data are available on the influence of processing, delay between processing and the use of sludge on the pathogenic risk. Producers and consumers will be more confident on the use of sludge if objective data are gained on risk. Most of the cases of cysticercosis (North America, United-Kingdom, Germany or Denmark) are related to poor human hygiene or accidental overflooding of sewage plants onto pastures. The standard application of sludge on pastures is apparently at low risk. This low risk does not mean that surveillance should cease since outbreaks of cysticercosis have been reported. Future investigations should concentrate on the most sustainable means of reducing risk (length of storage before use, composting, other treatments). (+info)
Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes.
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Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'. (+info)
Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system.
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By combination of denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA (PCR-DGGE), quinone profiling, and 16S rRNA-targeted fluorescence in situ hybridization (FISH), a polyphosphate-accumulating organism (PAO) responsible for phosphate (P)-removal was identified in activated sludge with high P-removal ability from a laboratory-scale anaerobic/aerobic continuous flow reactor. The DNA fragment from the most dense band on the DGGE gel was closely related to that of 'Candidatus Accumulibacter phosphatis' (beta-Proteobacteria). Quinone profiling also suggested the predominance of beta-Proteobacteria. FISH with a specific oligonucleotide probe designed for the sequence showed that the targeted bacterium was dominant in the activated sludge, and the accumulation and consumption of polyphosphate were observed by dual staining with 4',6-diamidino-2-phenylindole. The bacterium was concluded to be the responsible PAO in the reactor. However, when the P-removal ability per cell slightly decreased, the dominance of the PAO greatly diminished in the activated sludge. Such sludge might be dominated by other types of PAOs. (+info)
mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.
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mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouviere, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli. (+info)
Molecular characterization of astroviruses by reverse transcriptase PCR and sequence analysis: comparison of clinical and environmental isolates from South Africa.
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A comparative analysis was performed with 25 isolates of astroviruses (AstVs) detected in sewage sources and 22 concurrently identified clinical AstV isolates from the Tshwane (Pretoria) Metropolitan Area in South Africa. The samples and specimens were screened for AstVs by using an enzyme immunoassay and/or a reverse transcriptase PCR (RT-PCR) for the highly conserved untranslated region (3' end) of the genome. The RT-PCR results were confirmed by oligonucleotide probe dot blot hybridization. Viable viruses were propagated in cell cultures for amplification when a minimal specimen was available or indeterminate sequences were obtained. AstV strains were characterized by RT-PCR and partial sequence analysis of the capsid region. The presence of multiple human AstV (HAstV) types in a single sewage sample complicated identification of individual strains, and additional type-specific RT-PCR and sequence analyses of the capsid region were required for characterization. Amplification and characterization of one genotype from a sample, therefore, did not preclude the possibility that a sample harbored additional different genotypes. Genotype and sequence information obtained from AstVs in wastewater samples were compared to information obtained from AstV strains from human stools. HAstV type 1 (HAstV-1), as well as HAstV-3, -5, -6, and -8, were identified among the clinical isolates, and HAstV-1, -2, -3, -4, -5, -7, and -8 were identified among the environmental samples. Phylogenetic analysis demonstrated that HAstV-1, -3, -5, and -8, which were present in human stool and sewage samples, clustered together, indicating that these viruses are closely related. The concurrent presence of identical HAstV strains in wastewater samples and in hospitalized patients suggests that AstVs present in the environment pose a potential risk to communities in which fecally contaminated water is used for recreational and domestic purposes. (+info)
Monitoring gene expression in mixed microbial communities by using DNA microarrays.
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A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems. (+info)