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Whole-genome surveillance supports hazard profiling of Escherichia coli lineages in recycled water treatment systems.

UNLABELLED: The use of treated wastewater is increasingly important for sustainable water management under a changing climate, yet conventional monitoring based on Escherichia coli enumeration provides limited insight into strain diversity and associated public health hazards. Here, we applied longitudinal whole-genome sequencing (WGS) to 180 E. coli isolates collected across the treatment continuum of a recycled water facility, from influent to final effluent. Genomic analysis revealed extensive strain-level heterogeneity, comprising 88 sequence types across eight phylogroups, with greater diversity in influent than in treated effluent. Phylogenetic comparisons with contextual Australian genomes indicated clustering with strains associated with companion animals, wild birds, humans, and livestock, suggesting multiple potential source reservoirs rather than a single dominant origin, although source contributions were not definitive. Despite a >90% reduction in total E. coli loads, isolates recovered from upstream and downstream stages exhibited broadly comparable virulence factor and antimicrobial resistance gene (ARG) profiles, suggesting that, within the cultured isolate collection, reductions in abundance exceeded shifts in genomic composition. To assess operational relevance, we prototyped a genomics-informed hazard framework integrating virulence determinants, ARGs, plasmid-associated mobility, and reuse-specific exposure context. Using this framework, 92.8% of isolates were classified as low hazard, and 7.2% as moderate hazard, with no isolates meeting criteria for high or critical hazard classifications. These findings demonstrate that genomic profiling of indicator organisms can reveal population structure and hazard heterogeneity not captured by conventional enumeration alone, and can provide a practical basis for incorporating genomic information into hazard-informed monitoring of recycled water systems. IMPORTANCE: Routine recycled water monitoring relies largely on culture-based E. coli counts, which indicate regulatory compliance but provide limited insight into strain diversity, persistence, and genomic characteristics relevant to public health. Using longitudinal whole-genome sequencing, we show that genetically distinct E. coli lineages, including isolates carrying combinations of virulence and antimicrobial resistance determinants, can persist through advanced treatment despite substantial reductions in overall E. coli loads. While most isolates were classified as low genomic hazard and no high- or critical-hazard isolates were detected, these findings demonstrate that conventional enumeration alone cannot distinguish between genetically diverse lineages with differing hazard potential in highly treated systems. By integrating genomic data into a hazard classification framework, this study demonstrates an applied approach to contextualize E. coli detections and distinguish low-risk background populations from isolates with elevated genomic hazard profiles. This work supports the use of genomic profiling of indicator organisms to improve surveillance, inform treatment performance assessment, and enable more risk-based management of recycled water systems.

Escherichia coli

Mutagenicity and transformation by recycled water.

Increased use of contaminated water and long-range plans for the direct use of recycled water necessitate a careful assessment of the potential health effect on the population. Selected in vitro assays were used to evaluate the mutagenic and carcinogenic potential of a concentrated, recycled water sample. It was found that the concentrated water induced mutagenicity in hamster lung cells and cellular transformation in human embryonic lung fibroblasts. The use of in vitro analyses in conjunction with epidemiologic studies in determining the human risks of environmental carcinogens is discussed.

Animals

Phosphorylation of adenosine in aqueous solution by electric discharges.

Dehydration reactions involving condensing reagents in aqueous solution have been studied as models of chemical evolution, and one such reagent has been found to be produced in the supposed primitive Earth conditions. We assumed that the dehydration condensation in aqueous solution could occur if the condensing reagents, which appear to be produced by electric discharges in the gas phase, could be carried to the aqueous solution through the recycling of water washing the wall of the vessel. We present here an experimental study of the dehydration condensation between phosphate and adenosine in aqueous solution using a new discharge apparatus (Fig. 1) which simulates prebiotic chemical evolution. The apparatus was designed so that water recycles in a vessel containing a solution at a relatively low temperature (approximately 30 degrees C), achieved by showering water at 18 degrees C from above the vessel. This is a simulation of the recycling system of water on the Earth, depending on a large difference in temperature between the ocean surface and the sky.

Adenosine

Deposit and mobility of cadmium in a marsh-cove ecosystem and the relation to cadmium concentration in biota.

The study reported here presents the results of an investigation of a marsh-cove ecosystem heavily contaminated by cadmium. The most contaminated aquatic sediments were dredged in 1972-73, but the resuspension of the sediments and recycle of water from the dredge spoil resulted in reestablishment of a large contaminated sediment bed with concentrations very similar to those observed before dredging. The stability of the sediment concentrations and shallow depth of the cadmium in the sediments indicate that the deposit is relatively stable in agreement with the expectations based on the water chemistry of the system. Uptake does occur in both marsh and aquatic plants and all species of animals tested. Significantly elevated concentrations are observed compared to noncontaminated areas; however, edible portions of most fish do not appear to present a hazard. Crabs appear to present the most likely source of a hazard to humans. This potential hazard is still under investigation. The dredging removed about 5.5 MT of cadmium, about one-fourth of that originally estimated to be present, but twice that amount is found to be in the cove sediments 3 to 4 years after dredging. No appreciable improvement in the ecosystem has been made, and more careful consideration should be given to the need for decontamination and the method of removal of contaminated aquatic sediments in any future case.

Animals

Fin damage in captured and reared squids.

Fin damage was a major factor in the mortality of wild-caught squids kept in the laboratory. Infection of abraded fins by opportunistic bacterial pathogens impaired swimming and led to death. Serious skin abrasions were especially common in trawl-caught squids. Dipnets and jigs inflicted minimal trauma and were preferred for squid capture. Fin damage also occurred during transportation and during maintenance of squids in onshore tanks. A successful aquarium system with recycled sea water was used for squid maintenance. Hatchling, juvenile and adult loliginid squids remained healthy in closed-system aquaria for periods ranging from 1 to 16 weeks.

Animals

Unraveling the ecological success of Iodidimonas in a bioreactor treating oil and gas produced water.

UNLABELLED: Iodidimonas sp., a bacterium found in bioreactors treating oil and gas produced water as well as iodide-rich brines, has garnered attention for its unique ability to oxidize iodine. However, little is known about the metabolic capabilities that enable Iodidimonas sp. to thrive in certain unique ecological niches. In this study, we isolated, characterized, and sequenced three strains belonging to the Iodidimonas genus from the sludge of a membrane bioreactor used for produced water treatment. We investigated the genomic features of these isolates and compared them with the four publicly available isolate genomes from this genus, as well as a metagenome-assembled genome from the source bioreactor. Our Iodidimonas isolates had several genes associated with mitigating salinity, heavy metal, and organic compound stress, which likely help these bacteria to survive in produced water. Phenotyping tests revealed that while the isolates could utilize a wide variety of simple carbon substrates, they failed to degrade aliphatic or aromatic hydrocarbons, consistent with the lack of genes associated with common hydrocarbon degradation pathways in their genomes. We hypothesize that these microbes may lead a scavenging lifestyle in the bioreactor and similar iodide-rich brines. IMPORTANCE: Occupying a niche habitat and having few representative isolates, the genus Iodidimonas is a relatively understudied alphaproteobacterial group. Its ability to corrode pipes in iodine production facilities has economic implications, and its ability to generate potentially carcinogenic iodinated organic compounds during treatment of oil and gas produced water may cause environmental and health concerns with the recycling of treated water. Therefore, detailed characterization of the metabolic potential of the Iodidimonas isolates in this study both sheds light on their adaptation to the environmental conditions they inhabit and has environmental and economic significance.

Bioreactors

Mutagens in a river heavily polluted with paper recycling wastes: results of field and laboratory mutagen assays.

Paper recycling industries generate considerable quantities of waterborne wastes, and thus water pollution constitutes the greatest environmental problem associated with this industrial activity [Hunt and Franklin, 1973]. Generally the impact of this water pollution is considered in terms of aesthetic blight and deterioration of water quality. We present data that document another aspect of this pollution, the environmental contamination of an aquatic ecosystem with mutagenic materials. A natural population of the fern Osmunda regalis growing in a river heavily polluted with paper recycling wastes had a high incidence of chromosome mutations. This population was monitored for four years for the frequency of two-break chromosome mutations. These mutations were postzygotic in origin and suggested the presence of mutagens in the river water. The fern population is downstream from the outfalls of a paper recycling mill, which was discharging 13.3 X 10(6) liters of untreated paper recycling waste per day. In 1977 a waste-water-treatment facility was constructed to remove the solid waste previously discharged into the river. This facility generates 69,300 kg of solid waste daily, which is taken to a landfill. Periodic samples of this solid waste were collected from the waste-treatment facility in the summer of 1978, extracted with various solvents, and the extracts tested for mutagenic activity with the Salmonella/mammalian microsome mutagenicity test [Ames et al, 1975]. A majority of the solid waste samples contained mutagenic materials, but in all cases S-9 activation was required for mutagenic activity. The samples also were assayed with the soybean mitotic crossing-over assay [Vig, 1975]. Four out of six samples were positive. These results document the presence of mutagens in the solid waste generated by a paper recycling industry and the genetic impact of these mutagens on the local biota.

Chromosome Aberrations

Viruses in water.

Attention is drawn in this paper to the increasing problem of viral contamination of water and shellfish, particularly since growing demands for available water resources by a rising world population and expanding industry will make the recycling of wastewater almost inevitable in the future. The problem of eliminating viruses pathogenic for man from water is considered in the light of present water treatment procedures, which are often inadequate for that purpose. Man may be exposed to waterborne viruses through the consumption of contaminated water, shellfish, or crops, as a result of recreational activities involving water, or from aerosols following the spraying of crops with liquid wastes. Physical and chemical methods of eliminating viruses from water are discussed.

Humans

Three-dimensional porous nano-hydroxyapatite@gelatin composite as efficient adsorbent for uranyl ion removal from low-level radioactive wastewater.

The contamination of water resources by uranyl (UO22+) ions poses significant environmental and health risks, requiring the development of efficient and sustainable remediation strategies. Adsorption-based techniques have emerged as promising approaches in the field of UO22+ removal, but the design of cost-effective, high-capacity, and environmentally friendly adsorbents remains challenging. In this study, a three-dimensional porous nano-hydroxyapatite@gelatin (nHAP@Ge) composite was synthesized through glutaraldehyde cross-linking, combining the structural stability of Ge with the high uranium affinity of nHAP. The optimized nHAP@Ge, with a nHAP:Ge mass ratio of 1:0.5, exhibited exceptional UO22+ removal efficiency (97 %), along with high adsorption capacity (364.03 mg/g). Systematic characterizations using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) spectroscopy, and X-ray photoelectron spectroscopy (XPS) methods revealed that the porous structure and surface functional groups (-OH, Ca2+, and PO43-) of the material synergistically contributed to binding UO22+ species. Furthermore, the incorporation of nHAP into the Ge framework resulted in enhanced thermal stability while significantly improving the UO22+ adsorption performance. This work presents a scalable, eco-friendly, and recyclable strategy for the effective treatment of uranium-contaminated water, with potential applications in nuclear wastewater treatment and environmental remediation.

Adsorption

Observations on the biological roles of sulphatases.

Until recently little was known about the biological roles played by sulphatase enzymes, owing in part to the selection of assay substrates that were convenient but only removely related to the natural substrates. Once this was recognized the elucidation of function proceeded more rapidly. Microbial sulphatases appear to have roles to play in the nutrition of individual microorganisms whilst collectively they enable sulphur, returned to soils and waters in the form of sulphate esters, to be made available for recycling. In contrast, with one or two important exceptions, mammalian sulphatases are concerned, in association with other enzymes, with the turnover of macromolecules. Still defying understanding are the roles of sulphatases acting on adenosine 5'-phosphosulphate (APT) and 3'-phosphoadenosine 5'-phosphosulphate (PAPA). APS sulphatases have now been purified from ox-liver lysosomes and cytosol and from a strain of Comamonas terrigena. The lysosomal enzyme shows wide specificity and can hydrolyse ATP, ADP, FED and pyrophosphate. The cytosol enzyme is apparently specific and may be active only when cellular concentrations of ATP are low. The bacterial enzyme is also specific and has properties and a cellular localization that suggest the possibility of its involvement in sulphate transport.

Adenosine Monophosphate

[Bacterial destruction of synthetic organic flotation agents].

Microorganisms that use the flotation agent T-66 as the sole source of carbon have been isolated from soil enriched with this agent. A mixture of bacterial cultures belonging to the genus Pseudomonas (Ps. fluorescens, Ps. desmolyticum, Ps. rathonis, Ps. cyanoides viscosa, and Ps. aeruginosa) oxidized, after adaptation, at a high rate, components of the flotation agent T-66. Aeration of the medium accelerated the destruction. About 90% of the foltation agent components were oxidized within four months. The highest activity was observed during the first two months. The bacteria also decomposed, actively, oxidized kerosene and oxidized recycle stock added as the sole source of carbon to a mineral medium. Introduction of glucose to the medium accelerated destruction of oxidized recycle stock by the microorganisms, but inhibited destruction of oxidized kerosene.

Adaptation, Physiological

Purification of polyphosphoinositides by chromatography on immobilized neomycin.

The binding of polyphosphoinositides (phosphatidylinositol phosphate and phosphatidylinositol bisphosphate) to the antibiotic neomycin is utilized for the purification of these lipids. Neomycin is reductively coupled to reactive glass beads (Glycophase-CPG) and serves as the stationary phase in column chromatography. A total lipid extract is prepared from tissues with chloroform-methanol-KC1 or chloroform-methanol-HC1 and washed once with acidified methanol-water. After the addition of an equal volume of methanolic 200 mM ammonium acetate, the extract is directly applied to the column. All lipids but the polyphosphoinositides are removed from the column by rinsing with 150 mM ammonium acetate in chloroform-methanol-water. Increasing the salt concentration to 600 mM elutes phosphatidylinositol phosphate. While further increases in ionic strength are not sufficient for a quantitative removal of phosphatidylinositol bisphosphate, the lipid is completely eluted by the addition of either ammonia or HC1 to the solvent. The column can be recycled and used repeatedly.

Animals

Recycling of glucose by rat hepatocytes.

1. The metabolism of glucose labeled uniformly with 14C, and in positions 2, 3 and 5 with tritium by hepatocytes from fed and fasted rats were studied. Cells were incubated with glucose as sole substrate, or with glucose and a variety of glucose precursors, and uptake or production of glucose, and the utilization of the isotopes was determined. 2. There was no uptake of glucose at concentration of up to 15 mM, and net glucose synthesis in the presence of precursors. 14C was however recovered in CO2, lactate and amino acids, and tritium in water. Considerable incorporation into glycogen from 14C and 3H-labeled glucose occurred at high (above 20 mM) glucose concentrations. 3. The yield in water always exceeded that in 14C-labeled products. The yield in 3HOH from [2-3H] glucose exceeded that from [5-3H] glucose, and the latter was greater than from [3-3H] glucose. 4. Utilization of labeled glucose does not follow Michaelis-Menten kinetics. The fractional rate of uptake of 14C and tritium-labeled glucose increases with glucose concentration with a maximum at about 15 mM and then declines. 5. The effect of numerous gluconeogenic substrates on the isotope utilization and the 3H/14C ratio in glycogen was studied. The uptake of 14C was always depressed. Addition of lactate and dihydroxyacetone has little effect on the detritiation of [2-3H] glucose, but it is depressed by other substrates. The detritiation of [3-3H]-and[5-3H]glucose is depressed in gluconeogenesis, that from [3-3H]glucose usually more than from [5-3H]glucose. In the presence of lactate detritiation of [3-3H]glucose is about half that from [5-3H]glucose. 6. Equations to calculate the phosphorylation of glucose and fructose 6-phosphate in the presence of futile cycling between glucose and glucose 6-phosphate and fructose 6-phosphate and fructose 1,6-bisphosphate were derived. 7. The estimate of glucose phosphorylation requires determination of the specific activity of glucose 6-phosphate from [2-3H]glucose. It appears that futile cycling between glucose and glucose 6-phosphate is extensive in cells with a high glycogen content, but is low in cells from starved rats and nearly absent in those from diabetic animals. 8. The estimation of the phosphorylation of fructose 6-phosphate in the presence of cycling requires knowledge of the specific activities of fructose 6-phosphate and fructose 1,6-bisphosphate from [3-3H]glucose. At present there are no adqquate data to calculate phosphorylation and recycling of fructose 6-phosphate, but under some conditions the rate may be quite high.

Amino Acids

Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes.

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+, or Ni2+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than pS) to yield uridine 3'-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.

Enterobacter

Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats.

Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.

Adrenalectomy

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.

Animals