scholarly journals The Use of Ribosomal RNA as a Microbial Source Tracking Target Highlights the Assay Host-Specificity Requirement in Water Quality Assessments

2021 ◽  
Vol 12 ◽  
Author(s):  
Annastiina Rytkönen ◽  
Ananda Tiwari ◽  
Anna-Maria Hokajärvi ◽  
Sari Uusheimo ◽  
Asko Vepsäläinen ◽  
...  
Keyword(s):  
Surface Water ◽  
Ribosomal Rna ◽  
Water Samples ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Municipal Sewage ◽  
Source Tracking ◽  
Fecal Samples ◽  
Dna Template ◽  
Surface Water Samples

For microbial source tracking (MST), the 16S ribosomal RNA genes (rDNA) of host-specific bacteria and mitochondrial DNA (mtDNA) of animal species, known to cause fecal contamination of water, have been commonly used as molecular targets. However, low levels of contamination might remain undetected by using these DNA-based qPCR assays. The high copy numbers of ribosomal RNA (rRNA) could offer a solution for such applications of MST. This study compared the performance of eight MST assays: GenBac3 (general Bacteroidales), HF183 (human), BacCan (dog), Rum-2-Bac (ruminant), Pig-2-Bac (swine), Gull4 (gull), GFD, and Av4143 (birds) between rRNA-based and rDNA-based approaches. Three mtDNA-based approaches were tested: DogND5, SheepCytB, and HorseCytB. A total of 151 animal fecal samples and eight municipal sewage samples from four regions of Finland were collected for the marker evaluation. The usability of these markers was tested by using a total of 95 surface water samples with an unknown pollution load. Overall, the performance (specificity, sensitivity, and accuracy) of mtDNA-based assays was excellent (95–100%), but these markers were very seldom detected from the tested surface water samples. The rRNA template increased the sensitivity of assays in comparison to the rDNA template. All rRNA-based assays (except Av4143) had more than 80% sensitivity. In contrast, only half (HF183, Rum-2-Bac, Pig-2-Bac, and Gull4) of rDNA-based assays reached this value. For markers targeted to bird feces, the use of the rRNA-based assay increased or at least did not change the performance. Regarding specificity, all the assays had >95% specificity with a DNA template, except the BacCan assay (71%). While using the RNA template for the assays, HF183 and BacCan exhibited only a low level of specificity (54 and 55%, respectively). Further, the HF183 assay amplified from multiple non-targeted animal fecal samples with the RNA template and the marker showed cross-amplification with the DNA template as well. This study recommends using the rRNA-based approach for MST assays targeting bird fecal contamination. In the case of mammal-specific MST assays, the use of the rRNA template increases the sensitivity but may reduce the specificity and accuracy of the assay. The finding of increased sensitivity calls for a further need to develop better rRNA-based approaches to reach the required assay performance.

scholarly journals Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed

2014 ◽  
Vol 80 (8) ◽  
pp. 2328-2336 ◽  
Author(s):  
Natalie Prystajecky ◽  
Peter M. Huck ◽  
Hans Schreier ◽  
Judith L. Isaac-Renton
Keyword(s):  
Surface Water ◽  
Host Specificity ◽  
Water Samples ◽  
Environmental Protection Agency ◽  
Current Knowledge ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Genomic Sequencing ◽  
Content Type ◽  
Surface Water Samples

ABSTRACTKnowledge of host specificity, combined with genomic sequencing ofGiardiaandCryptosporidiumspp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period.Cryptosporidiumwas detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters.Giardiawas detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% ofCryptosporidiumsamples and 98% ofGiardiasamples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy.Cryptosporidiumgenotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared toGiardia, since 98% ofGiardiaisolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows thatCryptosporidiumsubtyping for MST purposes is superior to the use ofGiardiasubtyping, based on better detection limits forCryptosporidium-positive samples than forGiardia-positive samples and on greater host specificity amongCryptosporidiumspecies. These additional tools could be used for risk assessment in public health and watershed management decisions.

scholarly journals Evaluation of Two Library-Independent Microbial Source Tracking Methods To Identify Sources of Fecal Contamination in French Estuaries

2007 ◽  
Vol 73 (15) ◽  
pp. 4857-4866 ◽  
Author(s):  
Michèle Gourmelon ◽  
Marie Paule Caprais ◽  
Raphaël Ségura ◽  
Cécile Le Mennec ◽  
Solen Lozach ◽  
...  
Keyword(s):  
Water Samples ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rrna Gene ◽  
Specific Marker ◽  
Gene Markers ◽  
High Level ◽  
Bird Feces

ABSTRACT In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193′), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.

scholarly journals Synergistic Application of Molecular Markers and Community-Based Microbial Source Tracking Methods for Identification of Fecal Pollution in River Water During Dry and Wet Seasons

2021 ◽  
Vol 12 ◽  
Author(s):  
Hongxia Liang ◽  
Zhisheng Yu ◽  
Bobo Wang ◽  
Fabrice Ndayisenga ◽  
Ruyin Liu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Water Samples ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Small Sample ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Source Tracking ◽  
Community Based

It is important to track fecal sources from humans and animals that negatively influence the water quality of rural rivers and human health. In this study, microbial source tracking (MST) methods using molecular markers and the community-based FEAST (fast expectation–maximization microbial source tracking) program were synergistically applied to distinguish the fecal contributions of multiple sources in a rural river located in Beijing, China. The performance of eight markers were evaluated using 133 fecal samples based on real-time quantitative (qPCR) technique. Among them, six markers, including universal (BacUni), human-associated (HF183-1 and BacH), swine-associated (Pig-2-Bac), ruminant-associated (Rum-2-Bac), and avian-associated (AV4143) markers, performed well in the study. A total of 96 water samples from the river and outfalls showed a coordinated composition of fecal pollution, which revealed that outfall water might be a potential input of the Fsq River. In the FEAST program, bacterial 16S rRNA genes of 58 fecal and 12 water samples were sequenced to build the “source” library and “sink,” respectively. The relative contribution (<4.01% of sequence reads) of each source (i.e., human, swine, bovine, or sheep) was calculated based on simultaneous screening of the operational taxonomic units (OTUs) of sources and sinks, which indicated that community-based MST methods could be promising tools for identifying fecal sources from a more comprehensive perspective. Results of the qPCR assays indicated that fecal contamination from human was dominant during dry weather and that fecal sources from swine and ruminant were more prevalent in samples during the wet season than in those during the dry season, which were consistent with the findings predicted by the FEAST program using a very small sample size. Information from the study could be valuable for the development of improved regulation policies to reduce the levels of fecal contamination in rural rivers.

scholarly journals Evidence of Avian and Possum Fecal Contamination in Rainwater Tanks as Determined by Microbial Source Tracking Approaches

2016 ◽  
Vol 82 (14) ◽  
pp. 4379-4386 ◽  
Author(s):  
W. Ahmed ◽  
K. A. Hamilton ◽  
P. Gyawali ◽  
S. Toze ◽  
C. N. Haas
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Fecal Dna ◽  
Potential Health ◽  
Gene Copies ◽  
Tank Water ◽  
Rainwater Tanks

ABSTRACTAvian and possum fecal droppings may negatively impact roof-harvested rainwater (RHRW) water quality due to the presence of zoonotic pathogens. This study was aimed at evaluating the performance characteristics of a possum feces-associated (PSM) marker by screening 210 fecal and wastewater samples from possums (n= 20) and a range of nonpossum hosts (n= 190) in Southeast Queensland, Australia. The host sensitivity and specificity of the PSM marker were 0.90 and 0.95 (maximum value, 1.00), respectively. The mean concentrations of the GFD marker in possum fecal DNA samples (8.8 × 107gene copies per g of feces) were two orders of magnitude higher than those in the nonpossum fecal DNA samples (5.0 × 105gene copies per g of feces). The host sensitivity, specificity, and concentrations of the avian feces-associated GFD marker were reported in our recent study (W. Ahmed, V. J. Harwood, K. Nguyen, S. Young, K. Hamilton, and S. Toze, Water Res 88:613–622, 2016,http://dx.doi.org/10.1016/j.watres.2015.10.050). The utility of the GFD and PSM markers was evaluated by testing a large number of tank water samples (n= 134) from the Brisbane and Currumbin areas. GFD and PSM markers were detected in 39 of 134 (29%) and 11 of 134 (8%) tank water samples, respectively. The GFD marker concentrations in PCR-positive samples ranged from 3.7 × 102to 8.5 × 105gene copies per liter, whereas the concentrations of the PSM marker ranged from 2.0 × 103to 6.8 × 103gene copies per liter of water. The results of this study suggest the presence of fecal contamination in tank water samples from avian and possum hosts. This study has established an association between the degradation of microbial tank water quality and avian and possum feces. Based on the results, we recommend disinfection of tank water, especially for tanks designated for potable use.IMPORTANCEThe use of roof-harvested rainwater (RHRW) for domestic purposes is a globally accepted practice. The presence of pathogens in rainwater tanks has been reported by several studies, supporting the necessity for the management of potential health risks. The sources of fecal pollution in rainwater tanks are unknown. However, the application of microbial source tracking (MST) markers has the potential to identify the sources of fecal contamination in a rainwater tank. In this study, we provide evidence of avian and possum fecal contamination in tank water samples using molecular markers. This study established a potential link between the degradation of the microbial quality of tank water and avian and possum feces.

scholarly journals Microbial source tracking of private well water samples across at-risk regions in southern Ontario and analysis of traditional fecal indicator bacteria assays including culture and qPCR

2016 ◽  
Vol 14 (6) ◽  
pp. 1047-1058 ◽  
Author(s):  
Julia Krolik ◽  
Allison Maier ◽  
Shawna Thompson ◽  
Anna Majury
Keyword(s):  
At Risk ◽  
Water Samples ◽  
Rural Areas ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Well Water ◽  
Prevention Strategies ◽  
E Coli ◽  
Bacteriological Testing

Many people living in rural areas rely on privately owned wells as their primary source of drinking water. These water sources are at risk for fecal contamination of human, wildlife, and livestock origin. While traditional bacteriological testing involves culture-based methods, microbial source tracking (MST) assays present an opportunity to additionally determine the source of fecal contamination. This study investigated the main host sources of contamination in private well water samples with high levels of Escherichia coli (E. coli), using MST with human and multi-species specific markers. Fecal contamination of human origin was detected in approximately 50% of samples, indicating that current contamination prevention strategies require reconsideration. The relationship between cattle density and fecal contamination of bovine origin was investigated using a Bovine Bacteroidales specific MST assay. Regional variations of microbial sources were examined, and may inform local primary prevention strategies. Additionally, in order to assess MST and E. coli quantitative real time polymerase chain reaction (qPCR) assays as indicators of fecal contamination, these were compared to E. coli culture methods. Variation in results was observed across all assay methods investigated, suggesting the most appropriate routine bacteriological testing methodology cannot be determined without comparison to a method that directly detects the presence of fecal contamination.

scholarly journals Comparative Persistence of Subgroups of F-Specific RNA Phages in River Water

2013 ◽  
Vol 79 (15) ◽  
pp. 4564-4567 ◽  
Author(s):  
Yongheng Yang ◽  
Mansel W. Griffiths
Keyword(s):  
Surface Water ◽  
River Water ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Organic Substances ◽  
Misleading Information ◽  
Factors Affecting ◽  
Major Factors ◽  
Rna Phage

ABSTRACTF-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking.

scholarly journals Molecular Assays for Targeting Human and Bovine Enteric Viruses in Coastal Waters and Their Application for Library-Independent Source Tracking

2005 ◽  
Vol 71 (4) ◽  
pp. 2070-2078 ◽  
Author(s):  
Theng-Theng Fong ◽  
Dale W. Griffin ◽  
Erin K. Lipp
Keyword(s):  
Water Quality ◽  
Surface Water ◽  
Water Samples ◽  
Anthropogenic Activities ◽  
Fecal Contamination ◽  
Enteric Viruses ◽  
Binary Logistic Regression Analysis ◽  
Viral Pathogen ◽  
Molecular Assays ◽  
Surface Water Samples

ABSTRACT Rapid population growth and urban development along waterways and coastal areas have led to decreasing water quality. To examine the effects of upstream anthropogenic activities on microbiological water quality, methods for source-specific testing are required. In this study, molecular assays targeting human enteroviruses (HEV), bovine enteroviruses (BEV), and human adenoviruses (HAdV) were developed and used to identify major sources of fecal contamination in the lower Altamaha River, Georgia. Two-liter grab samples were collected monthly from five tidally influenced stations between July and December 2002. Samples were analyzed by reverse transcription- and nested-PCR. PCR results were confirmed by dot blot hybridization. Eleven and 17 of the 30 surface water samples tested positive for HAdV and HEV, respectively. Two-thirds of the samples tested positive for either HEV or HAdV, and the viruses occurred simultaneously in 26% of samples. BEV were detected in 11 of 30 surface water samples. Binary logistic regression analysis showed that the presence of both human and bovine enteric viruses was not significantly related to either fecal coliform or total coliform levels. The presence of these viruses was directly related to dissolved oxygen and streamflow but inversely related to water temperature, rainfall in the 30 days preceding sampling, and chlorophyll-a concentrations. The stringent host specificity of enteric viruses makes them good library-independent indicators for identification of water pollution sources. Viral pathogen detection by PCR is a highly sensitive and easy-to-use tool for rapid assessment of water quality and fecal contamination when public health risk characterization is not necessary.

Occurrence of 1,4-dioxane and MTBE in drinking water sources in Japan

2006 ◽  
Vol 6 (2) ◽  
pp. 47-53 ◽  
Author(s):  
D. Simazaki ◽  
M. Asami ◽  
T. Nishimura ◽  
S. Kunikane ◽  
T. Aizawa ◽  
...  
Keyword(s):  
Drinking Water ◽  
Surface Water ◽  
Water Samples ◽  
Quality Standards ◽  
Raw Water ◽  
Water Quality Standards ◽  
Butyl Ether ◽  
Water Treatment Plants ◽  
Standard Value ◽  
Surface Water Samples

Nationwide surveys of 1,4-dioxane and methyl-t-butyl ether (MTBE) levels in raw water used for the drinking water supply were conducted at 91 water treatment plants in Japan in 2001 and 2002, prior to the revision of the drinking water quality standards. 1,4-dioxane was widely and continuously detected in raw water samples and its occurrence was more frequent and its concentrations higher in groundwater than in surface water. However, its maximum concentration in raw water was much lower than its new standard value (50 μg/L), which was determined as a level of 10−5 excessive cancer risk to humans. Trace levels of MTBE were also detected in several surface water samples.

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