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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.

This study aimed to examine the drivers in shaping the occurrence and distribution of total viruses and picoplankton in tropical freshwater ecosystems. Flow cytometry was used to quantify the concentrations of total viruses, picoheterotrophs, picophytoplankton, and picocyanobacteria. Three land use patterns (urban-, agriculture- and parkland-dominated) were evaluated using ArcGIS. Significant correlations were observed between water-borne microbial targets and water quality parameters (0.175 ≤ |r| ≤ 0.441), nutrients (0.250 ≤ r ≤ 0.570) and land use factors (0.200 ≤ |r| ≤ 0.460). In particular, the concentrations of total viruses and picoheterotrophic cells were higher in catchments whereas the abundances of picophytoplankton and picocyanobacteria were higher in reservoirs. Total viruses and picoplankton had higher concentrations in urban- and agriculture-dominated areas, probably due to anthropogenic inputs and agricultural inputs, respectively. Although surface water is a complex matrix influenced by niche-based (i.e., physicochemical properties, nutrients, land use impact etc.) and neutral-based factors (i.e., ecological drift, dispersal and species), land use patterns could help to elucidate the occurrence and distribution of the total microbial community at the macroscopic level. Meanwhile, inter-correlations among viruses, picoplankton and picoheterotrophs (0.715 ≤ r ≤ 0.990) also substantiates their mutual interactions in influencing the microbial community. Furthermore, the relationships between total microbial cells and bacterial and viral indicators were also investigated. Concentrations of total viruses, picoplankton and picoheterotrophs were positively correlated with bacterial indicators (0.427 ≤ r ≤ 0.590) and viral indicators (0.201 ≤ r ≤ 0.563). These results indicated that faecal and viral contamination could contribute to the numbers of total viruses and bacteria.

This study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in Singapore and the geospatial distribution of the virome under different landuse patterns. Correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. Overall, the majority (65.5%) of the annotated virome belonged to bacteriophages. The percentage of Caudovirales was higher in reservoirs whereas the percentages of Dicistroviridae, Microviridae and Circoviridae were higher in tributaries. Reservoirs showed a higher Shannon-index virome diversity compared to upstream tributaries. Land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. Dicistroviridae and Microviridae were enriched in urbanized tributaries while Mimiviridae, Phycodnaviridae, Siphoviridae and Podoviridae were enriched in parkland reservoirs. Several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. In addition, the relative abundance of PMMoV (pepper mild mottle virus) sequences was significantly correlated with RT-qPCR measurements (0.588, Graphical abstract Image 1, Highlights • Land use is the main driver of the viral community structure in surface waters. • 66 viral families were detected and 66% of the annotated virome was bacteriophages. • Reservoirs showed a higher Shannon diversity (10.4) than upstream tributaries (8.4). • Sequences closely related to human picobirnavirus and cyclovirus were detected. • The relative abundance of PMMoV sequences was correlated with RT-qPCR measurements.

Fecal indicator bacteria, such as Escherichia coli (E.coli) and Enterococcus, have been widely used to indicate the presence of pathogens. However, the suitability of fecal indicator bacteria to represent health risks is still being challenged, particularly in tropical aquatic environments. The objective of this study is to understand the occurrence and prevalence of indicators and pathogens in areas with contrasting land use, as well as to identify the major correlations between indicators, pathogens and environmental parameters. The spatial and temporal variation of indicators and pathogens was studied to examine the distribution patterns for areas with different land use, and the impact of seasonal changes on microbial populations. A total of 234 water samples were sampled for two years from reservoirs and their tributaries, and tested for fecal indicator bacteria, coliphages, human specific markers, pathogenic bacteria and viruses. The prevalence of indicators and pathogens in reservoirs were generally low, while relatively high concentrations were observed in tributaries to varying degrees. Of the enteric viruses, norovirus GII was among the most prevalent and had the highest concentration. Although strong correlations were found between indicators, only relatively weak correlations were found between indicators and pathogens. The results in this study showed that none of the bacteria/phage indicators were universal predictors for pathogens. Inclusion of the alternative indicators, Methanobrevibacter smithii, Bacteroides and human polyomaviruses (HPyVs) to monitoring programs could help to determine whether the fecal source was human. The microbial distribution patterns allow the classification of sampling sites to different clusters and thus, help to identify sites which have poor water quality. This approach will be useful for water quality management to pinpoint factors that influence water quality and help to prioritize sites for restoration of water quality., Graphical abstract Image 1, Highlights • Norovirus GII was the most prevalent enteric viruses. • Land use classification was correlated to the distribution of pathogens/indicators. • Alternative indicators helped to improve the prediction of enteric viruses. • Cluster analysis identified sites with poor water quality.

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.

Enterococci have been recommended as suitable bacteria indicators for assessing the microbial quality of recreational waters. However, recent studies have shown that bacteria, including enterococci, are able to enter a viable but non-culturable (VBNC) state under environmentally stressed conditions, where they may remain undetected if culture-based methods are employed. To appreciate the extent of transformation of these cells in surface waters, a model Enterococcus organism, E. faecalis, was examined in laboratory controlled microcosms under different light and salinity conditions. Cells were detected by both standard culture-based and PMA-qPCR (propidium monoazide quantitative PCR) methods so that the VBNC cells could be enumerated. The decay rates from the culture based method (kc) and PMA-qPCR method (kp) were established for the different conditions. In general, the kC values (ranging from 0.0088 hr−1 to 0.9755 hr−1) were always higher than the kP values (0.0019 hr−1 to 0.2373 hr−1), implying that cells were able to retain their viability for much longer periods than what is shown by the culture-based method. In both cases, the k values generally showed an increasing trend with an increase in light irradiation, implying greater die-off with light. For freshwater microcosms, the kp values were 3–6 times lower than the kc values for different irradiation conditions, whereas for seawater the difference was up to 12 times, showing that E. faecalis adapts well to seawater. The kinetic data were used to develop models to describe the dynamics of VBNC formation in natural waters. At low light intensities (less than about 20 Wm-2), the proportion of VBNC cells was found to steadily increase to as high as 50%, even after 4 days. However, at higher light levels, this proportion was achieved more quickly (less than 5 h) but also diminished more rapidly. Hence, at high light levels, the percentage of VBNC cells is expected to be significant only for a few hours, whereas at low light levels, the VBNC cells can be expected to be present for a long period of time. These results have implications on the interpretation of microbial water quality data that are based on culture based methods.