Jean Pierre N.

A major challenge for assessment of water quality in tropical environments is the natural occurrence and potential growth of Fecal Indicator Bacteria (FIB). To gain a better understanding of the relationship between measured levels of FIB and the distribution of sewage-associated bacteria including potential pathogens in the tropics this study compared the abundance of FIB (Total coliforms and E. coli) and the Bacteroidales (HF183 marker) with bacterial community structure determined by… Show more

A major challenge for assessment of water quality in tropical environments is the natural occurrence and potential growth of Fecal Indicator Bacteria (FIB). To gain a better understanding of the relationship between measured levels of FIB and the distribution of sewage-associated bacteria including potential pathogens in the tropics this study compared the abundance of FIB (Total coliforms and E. coli) and the Bacteroidales (HF183 marker) with bacterial community structure determined by next-generation amplicon sequencing. Water was sampled twice over 6 months from 18 sites within a tropical urban catchment and reservoir, followed by extraction of DNA from microorganisms, and sequencing targeting the V3-V4 region of the 16S rRNA gene. Multivariate statistical analyses indicated that bacterial community composition (BCC) varied between reservoir and catchment, within catchment land-uses, and with E. coli concentration. Beta-regression indicated that the proportion of sequences from sewage-associated taxa (SAT) or pathogen-like sequences (PLS) were predicted most significantly by measured levels of E. coli (log MPN/100ml) (χ2>8.7; p<0.003). In addition, SAT were significantly predicted by log HF183 levels (χ2=13.1; p=0.0003) while PLS were not. Our study suggests that measurements of E. coli concentration could be useful in predicting samples enriched in sewage-associated and pathogen-like bacteria in tropical environments despite the potential for non-conservative behavior. Show less

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183… Show more

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively. Show less

Aims
The study goals were to determine the relationship between fecal indicator bacteria (FIB), the HF183 marker and land-use, and the phylogenetic diversity of HF183 marker sequences in a tropical urban watershed.

Methods and Results
Total coliforms, Escherichia coli, and HF183 were quantified in 81 samples categorized as undeveloped, residential, and horticultural from the Kranji Reservoir and Catchment in Singapore. Quantitative-PCR for HF183 followed by analysis of variance… Show more

Aims
The study goals were to determine the relationship between fecal indicator bacteria (FIB), the HF183 marker and land-use, and the phylogenetic diversity of HF183 marker sequences in a tropical urban watershed.

Methods and Results
Total coliforms, Escherichia coli, and HF183 were quantified in 81 samples categorized as undeveloped, residential, and horticultural from the Kranji Reservoir and Catchment in Singapore. Quantitative-PCR for HF183 followed by analysis of variance indicated that horticultural areas had significantly higher geometric-means for marker levels (4.3x104 HF183-GE 100ml−1) than non-horticultural areas (3.07x103 HF183-GE 100ml−1). E. coli and HF183 were moderately correlated in horticultural areas (R=0.59, p=0.0077), but not elsewhere in the catchment. Initial upstream surveys of candidate sources revealed elevated HF183 in a wastewater treatment effluent but not in aquaculture ponds. The HF183 marker was cloned, sequenced, and determined by phylogenetic analysis to match the original marker description.

Conclusion
We show that quantification of the HF183 marker is a useful tool for mapping the spatial distribution and potential sources of human sewage contamination in tropical environments such as Singapore.

Significance and Impact
A major challenge for assessment of water quality in tropical environments is the natural occurrence and non-conservative behavior of FIB. The HF183 marker has been employed in temperate environments as an alternative indicator for human sewage contamination. Our study supports use of the HF183 marker as an indicator for human sewage in Singapore and motivates further work to determine HF183 marker levels that correspond to public health risk in tropical environments. Show less