Present and future potential role of toxin-producing Synechococcus in the tropical region.

As anthropogenic induced temperature rises and nutrient loadings increase in fresh and brackish environments, the ecological function of the phytoplankton community is expected to favour the picocyanobacteria, of the genus Synechococcus. Synechococcus is already a ubiquitous cyanobacterium found in both freshwater and marine environments, notwithstanding that the toxigenic species still remains unexplored in many freshwaters. Their fast growth rate and their ability to produce toxins make Synechococcus a potential dominant player in harmful algal blooms under climate change scenarios. This study examines the responses of a novel toxin-producing Synechococcus (i.e., one belonging to a freshwater clade; the other belonging to a brackish clade) to environmental changes that reflect climate change effects. We conducted a series of controlled experiments under present and predicted future temperatures, as well as under various N and P nutrients loadings. Our findings highlight how Synechococcus can be altered by the differing reactions to increasing temperature and nutrients, which resulted in considerable variations in cell abundance, growth rate, death rate, cellular stoichiometry and toxin production. Synechococcus had the highest growth observed at 28 °C, and further increases in temperature resulted in a decline for both fresh and brackish waters. Cellular stoichiometry was also altered, where more nitrogen (N) per cell was required, and the plasticity of N:P was more severe for the brackish clade. However, Synechococcus become more toxic under future scenario. Anatoxin-a (ATX) saw the greatest spike when temperature was at 34 °C especially under P-enrichment conditions. In contrast, Cylindrospermopsin (CYN) was promoted at the lowest tested temperature (25 °C) and under N-limitation. Overall, both temperature and external nutrients are the dominant control over Synechococcus toxins production. A model was also created to assess Synechococcus toxicity to zooplankton grazing. Zooplankton grazing was reduced by two folds under nutrient limitation, but temperature accounted for very insignificant change.

Assessment of Human Health Risks in Tropical Environmental Waters with Microbial Source Tracking Markers.

Human specific microbial source tracking (MST) markers which are highly specific to human waste contamination offer the advantage of better association with human pathogens than traditional microbial indicators. However, the performance of these MST markers may vary across different geographical regions. The magnitude of MST markers also plays an important role in interpreting the health risks. This study aims to (i) validate the specificity and sensitivity of human markers for tropical urban catchments; (ii) identify the threshold concentrations of MST markers, i.e. human polyomaviruses (HPyVs), Bacteroides thetaiotaomicron (B. theta) and Methanobrevibacter smithii (M. smithii), that correspond to the acceptable gastrointestinal (GI) illness risks associated with swimming using the QMRA approach; and (iii) validate the threshold concentrations of MST markers using the surveillance data obtained from the tropical urban environment. Among the three MST markers, HPyVs showed the highest specificity (100%) to sewage samples, followed by M. smithii (97%) and B. theta (90%). All MST markers showed 100% sensitivity towards sewage contamination, with B. theta present in highest abundance in sewage, followed by HPyVs and M. smithii. This study demonstrates a risk-based framework to identify the threshold concentrations of MST markers associated with GI illness risks in environmental waters by considering two main influencing factors (i.e. decay and dilution factors). This study successfully validated the B. theta threshold concentration range (581 to 8073 GC/100 mL) with field data (370 to 6500 GC/100 mL) in estimating GI illness risks with an Enterococcus model. Field data showed that the MST markers at threshold concentrations were able to classify the safe level in more than 83% of the samples, according to GI illness risks from Enterococcus and adenovirus. The study also highlighted the lack of associations between MST markers and GI illness risks from norovirus. With comprehensive information on specificity, sensitivity and threshold concentrations of MST markers, increasing confidence can be placed on identifying human source contamination and evaluating the health risks posed in environmental waters in Singapore.

Using Pseudomonas aeruginosa PAO1 to evaluate hydrogen peroxide as a biofouling control agent in membrane treatment systems.

Hydrogen peroxide (H2 O2 ) is widely used in water treatment for biofouling control and, in conjunction with catalysts, as a powerful oxidant for contaminant destruction. H2 O2 could potentially serve as an antifouling agent in reverse osmosis systems in lieu of chlorine-based disinfectants. The dependence of the biocidal efficiency of H2 O2 on cell density, temperature and H2 O2 concentration by determining the growth, attachment and viability of the model bacterium Pseudomonas aeruginosa PAO1 was studied. For controlling growth of planktonic PAO1 cells, the minimally required H2 O2 concentration depends on the cell density and temperature. The effect of H2 O2 to remove the existing biofilm was found to be effective in the presence of a high concentration bicarbonate (8·4 g l-1 ), which forms peroxymonocarbonate, a strong oxidant and disinfectant. Treatment with H2 O2 -bicarbonate reduced the density of live PAO1 cells, removed extracellular polymeric substances and lowered the average biofilm thickness while maintaining the integrity of the membrane, suggesting that this type of treatment may be a suitable 'in-place-cleaning' procedure for biofouled membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: H2 O2 is evaluated as a potential replacement for chlorine to control biofouling in membrane-based water treatment systems. The biocidal efficacy of H2 O2 was evaluated as a function of H2 O2 concentration, cell density and temperature using the model organism Pseudomonas aeruginosa PAO1. Results demonstrated that at low temperatures and low cell densities, bacterial growth and membrane biofouling can be prevented by low H2 O2 concentrations, and existing biofilms could be removed by H2 O2 -bicarbonate mixtures. Findings suggested that H2 O2 could be used as a low cost agent for prevention and controlling biofouling in reverse osmosis applications.

Risk assessment of noroviruses and human adenoviruses in recreational surface waters.

In the interest of public health and safety, this study aimed to quantify risks associated with the presence of noroviruses (NoV) and human adenoviruses (HAdV) in an urban catchment area in Singapore. Enteric viruses were quantified using QPCR. NoV were more prevalent in water samples than HAdV, and presented higher associated illness risks across all exposure scenarios. For primary contact recreation of adults and children, mean probability of illness were 0.0061 and 0.0089 for NoV, and 0.0028 and 0.0048 for HAdV. For secondary contact recreation, mean probability of illness were 0.0016 for NoV and 0.00068 for HAdV. Therefore, owing to their prevalence and associated risks, NoV are better suited as reference pathogens in recreational waters in Singapore.

Evaluation of FRNA coliphages as indicators of human enteric viruses in a tropical urban freshwater catchment.

This study aimed to evaluate the relationship between FRNA coliphages (FRNA GI to GIV) and human enteric viruses (human adenoviruses, HAdV, astroviruses, AstV, noroviruses, NoV, and rotaviruses, RoV) in a tropical urban freshwater catchment. Positive associations between human-specific coliphages and human viral pathogens substantiate their use as viral indicators and in microbial source tracking. Reverse transcription qPCR was used to measure the concentrations of viruses and FRNA coliphages in concentrated water samples. Environmental water samples were also analyzed for male-specific (F+) and somatic (Som) coliphages using plaque assay. The most abundant enteric virus was NoV (55%) followed by HAdV (33%), RoV (33%), and AstV (23%), while the most abundant FRNA genogroup was GI (85%) followed by GII (48%), GIV (8%) and GIII (7%). Concentrations of human-specific coliphages FRNA GII were positively correlated with NoV, HAdV, RoV, AstV, F+ and Som (τ = 0.5 to 0.3, P < 0.05) while concentrations of animal-specific coliphages FRNA GI were negatively correlated with HAdV and RoV (τ = -0.2, P < 0.05). This study demonstrates statistical relationships between human-specific coliphages and a suite of human enteric viruses in the environment.

Alternative fecal indicators and their empirical relationships with enteric viruses, Salmonella enterica, and Pseudomonas aeruginosa in surface waters of a tropical urban catchment.

The suitability of traditional microbial indicators (i.e., Escherichia coli and enterococci) has been challenged due to the lack of correlation with pathogens and evidence of possible regrowth in the natural environment. In this study, the relationships between alternative microbial indicators of potential human fecal contamination (Bacteroides thetaiotaomicron, Methanobrevibacter smithii, human polyomaviruses [HPyVs], and F+ and somatic coliphages) and pathogens (Salmonella spp., Pseudomonas aeruginosa, rotavirus, astrovirus, norovirus GI, norovirus GII, and adenovirus) were compared with those of traditional microbial indicators, as well as environmental parameters (temperature, conductivity, salinity, pH, dissolved oxygen, total organic carbon, total suspended solids, turbidity, total nitrogen, and total phosphorus). Water samples were collected from surface waters of urban catchments in Singapore. Salmonella and P. aeruginosa had significant positive correlations with most of the microbial indicators, especially E. coli and enterococci. Norovirus GII showed moderately strong positive correlations with most of the microbial indicators, except for HPyVs and coliphages. In general, high geometric means and significant correlations between human-specific markers and pathogens suggest the possibility of sewage contamination in some areas. The simultaneous detection of human-specific markers (i.e., B. thetaiotaomicron, M. smithii, and HPyVs) with E. coli and enterococcus supports the likelihood of recent fecal contamination, since the human-specific markers are unable to regrow in natural surface waters. Multiple-linear-regression results further confirm that the inclusion of M. smithii and HPyVs, together with traditional indicators, would better predict the occurrence of pathogens. Further study is needed to determine the applicability of such models to different geographical locations and environmental conditions.

Surveillance of enteric viruses and coliphages in a tropical urban catchment.

An assessment of the occurrence and concentration of enteric viruses and coliphages was carried out in highly urbanized catchment waters in the tropical city-state of Singapore. Target enteric viruses in this study were noroviruses, adenoviruses, astroviruses and rotaviruses. In total, 65 water samples were collected from canals and the reservoir of the Marina catchment on a monthly basis over a period of a year. Quantitative PCR (qPCR) and single agar layer plaque assay (SAL) were used to enumerate target enteric viruses and coliphages in water samples, respectively. The most prevalent pathogen were noroviruses, detected in 37 samples (57%), particularly norovirus genogroup II (48%), with a mean concentration of 3.7 × 10(2) gene copies per liter. Rotavirus was the second most prevalent virus (40%) with a mean concentration of 2.5 × 10(2) GC/L. The mean concentrations of somatic and male-specific coliphages were 2.2 × 10(2) and 1.1 × 10(2) PFU/100 ml, respectively. The occurrence and concentration of each target virus and the ratio of somatic to male-specific coliphages varied at different sampling sites in the catchment. For sampling sites with higher frequency of occurrence and concentration of viruses, the ratio of somatic to male-specific coliphages was generally much lower than other sampling sites with lower incidences of enteric viruses. Overall, higher statistical correlation was observed between target enteric viruses than between enteric viruses and coliphages. However, male-specific coliphages were positively correlated with norovirus concentrations. A multi-level integrated surveillance system, which comprises the monitoring of bacterial indicators, coliphages and selected enteric viruses, could help to meet recreational and surface water quality criteria in a complex urbanized catchment.

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments.

AIMS: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. METHODS AND RESULTS: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co-circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. CONCLUSIONS: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas.

Environmental surveillance and molecular characterization of human enteric viruses in tropical urban wastewaters.

AIMS: To study the prevalence and genotypes of waterborne pathogenic viruses in urban wastewaters in the tropical region. METHODS AND RESULTS: Viruses in wastewaters collected at three water reclamation plants in Singapore were studied by molecular methods. Over a 6-month sampling period, adenoviruses, astroviruses and both norovirus genogroups I (GI) and II (GII) were detected in 100% of the sewage and secondary effluent. Enteroviruses and hepatitis A viruses (HAV) were found in 94 and 78% of sewage, and 89 and 28% of secondary effluent, respectively. By using quantitative real-time PCR, estimated concentrations of astrovirus in the sewage were 1-2 orders of magnitude higher than those for adenovirus, noroviruses GI and GII. Genotyping of environmental isolates revealed multiple genotypes of GI and GII noroviruses. Coxsackieviruses A, astrovirus type 1 and adenovirus type 41 were prevalent. Norovirus GII/4 and coxsackievirus A24 isolates in wastewaters were closely related to respective outbreak strains isolated previously in Singapore. CONCLUSIONS: This study showed the widespread occurrence of all tested enteric virus groups in urban wastewaters. Genetic diversity of astroviruses, enteroviruses and noroviruses in the tropical region was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence and great genetic diversity of human enteric viruses in urban wastewaters strongly supports the need of further comprehensive studies for evaluating the public health risk associated with viral pathogens in water environments.