Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

ABSTRACT Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

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Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

Applied and Environmental Microbiology ◽

10.1128/aem.02032-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 7067-7077 ◽

Cited By ~ 42

Author(s):

W. Ahmed ◽

C. Staley ◽

M. J. Sadowsky ◽

P. Gyawali ◽

J. P. S. Sidhu ◽

...

Keyword(s):

Molecular Markers ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Water Samples ◽

High Throughput Sequencing ◽

Community Analysis ◽

Fecal Pollution ◽

Rrna Gene ◽

Potential Sources

ABSTRACTIn this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

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THE 16S rRNA GENE MARKER-BASED MOLECULAR IDENTIFICATION OF CULTURABLE COLIFORM BACTERIA ISOLATED FROM FORAMINIFERA CALCARINA DERIVED FROM PRAMUKA ISLAND WATERS, THE SERIBU ISLAND DISTRICT, JAKARTA PROVINCE

Jurnal Harpodon Borneo ◽

10.35334/harpodon.v13i1.1404 ◽

2020 ◽

Vol 13 (1) ◽

pp. 10-18

Author(s):

Mochamad Untung Kurnia - Agung ◽

Agus Tri Askar ◽

Yuli Andriani ◽

Lintang Permatasari Yuliadi

Keyword(s):

Coral Reef ◽

16S Rrna ◽

16S Rrna Gene ◽

Molecular Identification ◽

Bay Of Bengal ◽

Coliform Bacteria ◽

Gene Marker ◽

Rrna Gene ◽

Gene Markers ◽

The 16S Rrna Gene

Contamination of coliform bacteria in benthic foraminifera has been reported due to pollution of organic wastes in the aquatic environment around coral reef ecosystems and this event was known to interfere the process of foraminifera shell formation which in turn resulted the disruption of the role of foraminifera in the process of formation of coral reef bottom sediments. The aim of this research is to identify the isolates of culturable coliform bacteria that contaminate foraminifera Calcarina species isolated from the waters of the Pramuka Island, the Seribu Island district, Jakarta Province using the 16S rRNA gene markers. Foraminifera sampling was carried out in the waters of Pramuka Island, the Seribu Island district, Jakarta Province in 5 (five) stations, while the process of bacterial isolation and molecular identification were carried out at the Laboratory of Microbiology and Molecular Biotechnology (MICROMOL), Faculty of Fisheries and Marine Sciences (FPIK), University Padjadjaran. Molecular identification was carried out using the Polymerase Chain Reaction (PCR) method based on the 16S rRNA gene markers. Sequencing is done by sending PCR results to 1st Base, sequencing service company, in Singapore and then, the aligning of sequencing results with databases in genBank was done using the Basic Local Alignment Search Tool (BLASTTM) program available on the National Center for Biotechnology Information (NCBI) website. The results of 16S rRNA gene amplification from the five isolates produced amplicons of ± 1400 bp length with concentrations ranging from 157.5 µg / mL-230 µg / mL and with a purity ratio ranging from 1.477-1.769. While the results of BLAST and phylogenetic analysis showed that the five isolates were closely related to the isolate Eschericia coli strain inspire99 (Acc No. JQ315935.1), which was isolated from the waters of the Bay of Bengal, India. These results also indicate the existence of ecological connectivity between the waters of the Bay of Bengal in India and the waters of Pramuka Island in Indonesia.

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Phylogeny and affiliation of European Anthomyzidae (Diptera) based on mitochondrial 12S and 16S rRNA

Zootaxa ◽

10.11646/zootaxa.2054.1.3 ◽

2009 ◽

Vol 2054 (1) ◽

pp. 49-58 ◽

Cited By ~ 8

Author(s):

JINDŘICH ROHÁČEK ◽

ANDREA TÓTHOVÁ ◽

JAROMÍR VAŇHARA

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bayesian Analysis ◽

Phylogenetic Relationships ◽

Posterior Probability ◽

Morphological Data ◽

Rrna Gene ◽

Gene Markers ◽

The Family ◽

Species Groups

The phylogenetic Bayesian analysis of combined mitochondrial 12S and 16S rRNA gene markers in European representatives of Anthomyzidae supported the monophyly of the family, confirmed the validity of the genera and provided a resolution of the generic affiliation of some aberrant species. The interrelationships of most genera have not been resolved definitively due to insufficient posterior probability values. Despite this, the clade clustering the genus Anthomyza Fallén, 1810, Fungomyza Roháček, 1999 and the Anthomyza socculata group is considered to reflect the real phylogenetic relationships of these taxa as it is also supported by morphological data. The relationships within the genus Anthomyza are resolved and monophyly of its species-groups confirmed. Within the genus Stiphrosoma Czerny, 1928, S. fissum Roháček, 1996, a species of unknown relationships, was found to be most closely allied to S. sabulosum (Haliday, 1837). Based on the above analysis a removal of the aberrant species Paranthomyza caricis Roháček, 1999 and Anthomyza socculata (Zetterstedt, 1847) from genera to which they were formerly affiliated, is suggested.

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Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2651-2656.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2651-2656 ◽

Cited By ~ 39

Author(s):

Yolanda Nebra ◽

Xavier Bonjoch ◽

Anicet R. Blanch

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Hybridization ◽

Fecal Pollution ◽

Rrna Gene ◽

Human Origin ◽

Threshold Detection ◽

Specific Protocol ◽

Fecal Water ◽

Potential Use

ABSTRACT A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.

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