Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents.

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents.

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Sanitary inspection of wells using risk-of-contamination scoring indicates a high predictive ability for bacterial faecal pollution in the peri-urban tropical lowlands of Dar es Salaam, Tanzania.

Sanitary inspection of wells was performed according to World Health Organization (WHO) procedures using risk-of-contamination (ROC) scoring in the peri-urban tropical lowlands of Dar es Salaam, Tanzania. The ROC was assessed for its capacity to predict bacterial faecal pollution in the investigated well water. The analysis was based on a selection of wells representing environments with low to high presumptive faecal pollution risk and a multi-parametric data set of bacterial indicators, generating a comprehensive picture of the level and characteristics of faecal pollution (such as vegetative Escherichia coli cells, Clostridium perfringens spores and human-associated sorbitol fermenting Bifidobacteria). ROC scoring demonstrated a remarkable ability to predict bacterial faecal pollution levels in the investigated well water (e.g. 87% of E. coli concentration variations were predicted by ROC scoring). Physicochemical characteristics of the wells were not reflected by the ROC scores. Our results indicate that ROC scoring is a useful tool for supporting health-related well water management in urban and suburban areas of tropical, developing countries. The outcome of this study is discussed in the context of previously published results, and future directions are suggested.

Sorbitol-fermenting Bifidobacteria are indicators of very recent human faecal pollution in streams and groundwater habitats in urban tropical lowlands.

Sorbitol-fermenting Bifidobacteria (SFB) proved to be an excellent indicator of very recent human faecal pollution (hours to days) in the investigated tropical stream and groundwater habitats. SFB were recovered from human faeces and sources potentially contaminated with human excreta. SFB were undetectable in animal faeces and environmental samples not contaminated with human faeces. Microcosm studies demonstrated a rapid die-off rate in groundwater (T90 value 0.6 days) and stream water (T90 value 0.9-1.7 days). Discrimination sensitivity analysis, including E. coli, faecal coliforms, total coliforms and Clostridium perfringens spores, revealed high ability of SFB to distinguish differing levels of faecal pollution especially for streams although high background levels of interfering bacteria can complicate its recovery on the used medium. Due to its faster die-off, as compared to many waterborne pathogens, SFB cannot replace microbiological standard parameters for routine water quality monitoring but it is highly recommendable as a specific and complementary tool when human faecal pollution has to be localized or verified. Because of its exclusive faecal origin and human specificity it seems also worthwhile to include SFB in future risk evaluation studies at tropical water resources in order to evaluate under which situations risks of infection may be indicated.

Use of faecal pollution indicators to estimate pathogen die off conditions in source separated faeces in Kathmandu Valley, Nepal.

As the introduction and promotion of dehydrating toilets progresses, the safety of handling and reuse of their biosolids remains a question. A detailed study to understand the storage conditions and the fate of selected faecal indicators was conducted on four urine diverting dehydrating toilet units, using ash as a major additive, in Kathmandu Valley, Nepal. Presumptive Escherichia coli, total coliforms, enterococci and different fractions of Clostridium perfringens were investigated under field storage conditions. In addition, chemo-physical and chemical (carbon, nitrogen, phosphorous content) parameters were investigated. Observed temperature was low in all the four toilets with a median of 24.0 degrees C, which was in the same range as the ambient temperature. pH was below the desired range of >9 and moisture level was very high (>60%). No single factor of the studied chemo-physical and chemical parameters could be found by statistical analysis to have accounted for the reduction of the indicators in any of the toilets. By time series analysis of the investigated strata in the faecal heaps (n=96), the determined reduction rate showed increasing persistence characteristics for E. coli, coliforms and enterococci with respective average log(10) reduction of -0.4, -0.3 and -0.2 per month (p<0.001). No significant reduction was observed for the different fractions of C. perfringens determined for the non-pasteurised and pasteurised fraction at 60 degrees C and 85 degrees C. 72% of randomly selected and analysed samples (n=36) were found to contain helminthes eggs. The used 6 months storage time did not prove sufficient to reach appropriate safety levels for handling and reuse of the biosolids.

Contrasting occurrence of Chromobacterium violaceum in tropical drinking water springs of Uganda.

Occurrence of Chromobacterium violaceum in six protected drinking water springs in Uganda was investigated. C. violaceum showed a contrasting occurrence, which was independent of human impact as assessed by faecal pollution indicators. It was isolated from two springs (S1 and S2) that were located close to each other (3 km) but not in the rest. In S1 C. violaceum was continuously detected, in concentrations ranging from 6 to 270 cfu 100 ml(-1), while in S2 it was detected on only one sampling occasion. C. violaceum was never detected in the investigated upper soil layers (down to 15 cm) in the immediate surroundings (50 m radius) of the springs, despite continued isolation of faecal indicators. The results of the study indicate that C. violaceum may not be ubiquitous in spring water, but could occur in significant numbers in particular potable groundwaters as an autochthonous member.

Discrimination efficacy of fecal pollution detection in different aquatic habitats of a high-altitude tropical country, using presumptive coliforms, Escherichia coli, and Clostridium perfringens spores.

The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa.