scholarly journals Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

2014 ◽  
Vol 81 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Vikram Kapoor ◽  
Tarja Pitkänen ◽  
Hodon Ryu ◽  
Michael Elk ◽  
David Wendell ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Fecal Pollution ◽  
Detection Sensitivity ◽  
Rrna Gene ◽  
Fecal Bacteria ◽  
Combined Sewer Overflows ◽  
Fecal Indicator ◽  
Content Type ◽  
Detection Frequency ◽  
Human Specific

ABSTRACTThe identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci,Enterococcus faecalis, andEnterococcus faeciummarkers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specificBacteroidalesmarkers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specificBacteroidalesmarkers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

scholarly journals Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor

2011 ◽  
Vol 77 (19) ◽  
pp. 6972-6981 ◽  
Author(s):  
Ryan J. Newton ◽  
Jessica L. VandeWalle ◽  
Mark A. Borchardt ◽  
Marc H. Gorelick ◽  
Sandra L. McLellan
Keyword(s):  
Sequence Analysis ◽  
Genetic Marker ◽  
Microbial Community Composition ◽  
Fold Increase ◽  
Fecal Pollution ◽  
Plate Count ◽  
Rrna Gene ◽  
Fecal Indicators ◽  
Fecal Indicator ◽  
Content Type

ABSTRACTThe complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within theLachnospiraceaefamily, which was closely related to the genusBlautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for humanBacteroidales(based on the HF183 genetic marker), totalBacteroidalesspp., and enterococci and the conventionalEscherichia coliand enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and humanBacteroidalesincreased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.

scholarly journals Correlation of Quantitative PCR for a Poultry-SpecificBrevibacteriumMarker Gene with Bacterial and Chemical Indicators of Water Pollution in a Watershed Impacted by Land Application of Poultry Litter

2011 ◽  
Vol 77 (6) ◽  
pp. 2094-2102 ◽  
Author(s):  
Jennifer L. Weidhaas ◽  
Tamzen W. Macbeth ◽  
Roger L. Olsen ◽  
Valerie J. Harwood
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Marker Gene ◽  
Poultry Litter ◽  
Land Application ◽  
Fecal Pollution ◽  
Health Concern ◽  
Rrna Gene ◽  
Fecal Indicator ◽  
The Impact

ABSTRACTThe impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene ofBrevibacteriumsp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations ofEscherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

scholarly journals Quantitative PCR for Genetic Markers of Human Fecal Pollution

2009 ◽  
Vol 75 (17) ◽  
pp. 5507-5513 ◽  
Author(s):  
Orin C. Shanks ◽  
Catherine A. Kelty ◽  
Mano Sivaganesan ◽  
Manju Varma ◽  
Richard A. Haugland
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Quantitative Methods ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Target Dna ◽  
Pcr Assays ◽  
Wastewater Samples ◽  
Human Specific

ABSTRACT Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 � 106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

scholarly journals Human-Associated Lachnospiraceae Genetic Markers Improve Detection of Fecal Pollution Sources in Urban Waters

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Shuchen Feng ◽  
Melinda Bootsma ◽  
Sandra L. McLellan
Keyword(s):  
Urban Areas ◽  
Human Microbiome ◽  
Cross Reactivity ◽  
Fecal Pollution ◽  
Pollution Sources ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Fecal Indicator ◽  
Content Type ◽  
Nonspecific Amplification

ABSTRACT The human microbiome contains many organisms that could potentially be used as indicators of human fecal pollution. Here we report the development of two novel human-associated genetic marker assays that target organisms within the family Lachnospiraceae . Next-generation sequencing of the V6 region of the 16S rRNA gene from sewage and animal stool samples identified 40 human-associated marker candidates with a robust signal in sewage and low or no occurrence in samples from nonhuman hosts. Two were chosen for quantitative PCR (qPCR) assay development using longer sequences (the V2 to V9 regions) generated from clone libraries. Validation of these assays with these markers, designated Lachno3 and Lachno12, was performed using fecal samples ( n = 55) from cat, dog, pig, cow, deer, and gull sources, and the results were compared with those of established host-associated assays (the Lachno2 marker and two human Bacteroides markers, the HB and HF183/BacR287). Each of the established assays cross-reacted with samples from at least one other animal species, including animals common in urban areas. The Lachno3 and Lachno12 markers were primarily human associated; however, the Lachno12 marker demonstrated low levels of cross-reactivity with samples from select cows and nonspecific amplification with samples from pigs. This limitation may not be problematic when testing urban waters. These novel markers resolved ambiguous results from previous investigations of stormwater-impacted waters, demonstrating their utility. The complexity of the microbiome in humans and animals suggests that no single organism is strictly specific to humans, and the use of multiple complementary markers in combination will provide the highest resolution and specificity for assessing fecal pollution sources. IMPORTANCE Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.

scholarly journals Multitiered Approach Using Quantitative PCR To Track Sources of Fecal Pollution Affecting Santa Monica Bay, California

2006 ◽  
Vol 72 (2) ◽  
pp. 1604-1612 ◽  
Author(s):  
Rachel T. Noble ◽  
John F. Griffith ◽  
A. Denene Blackwood ◽  
Jed A. Fuhrman ◽  
Jason B. Gregory ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Fecal Indicator Bacteria ◽  
Fecal Contamination ◽  
Fecal Pollution ◽  
Indicator Bacteria ◽  
Fecal Indicator ◽  
E Coli ◽  
Santa Monica Bay ◽  
Santa Monica ◽  
Human Specific

ABSTRACT The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 109 to 1010 cells h−1 and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.

scholarly journals Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

2012 ◽  
Vol 78 (12) ◽  
pp. 4338-4345 ◽  
Author(s):  
Hodon Ryu ◽  
Jingrang Lu ◽  
Jason Vogel ◽  
Michael Elk ◽  
Felipe Chávez-Ramírez ◽  
...  
Keyword(s):  
Host Specificity ◽  
Quantitative Pcr ◽  
Water Samples ◽  
False Negative ◽  
Environmental Water ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Content Type ◽  
Sandhill Crane

ABSTRACTWhile the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous toBacteroidetesandClostridia. The Crane1 marker targeted a dominant clade of unclassifiedLactobacillalessequences closely related toCatellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e.,n= 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n= 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n= 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.

scholarly journals Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

2013 ◽  
Vol 80 (2) ◽  
pp. 757-765 ◽  
Author(s):  
Amber M. Koskey ◽  
Jenny C. Fisher ◽  
Mary F. Traudt ◽  
Ryan J. Newton ◽  
Sandra L. McLellan
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Sequence Comparisons ◽  
Fecal Indicator ◽  
Fecal Samples ◽  
Content Type

ABSTRACTGulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such asEscherichia coliand enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB.EnterococcaceaeandEnterobacteriaceaewere the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealedCatellicoccus marimammaliumas the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcusindoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified asEnterococcusspp., 1.2% were identified asStreptococcusspp., and none were identified asC. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions ofC. marimammalium16S rRNA gene sequences (>50-fold) relative to typical mEI culturableEnterococcusspp.C. marimammaliumtherefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.

scholarly journals Correlation between Quantitative PCR and Culture-Based Methods for Measuring Enterococcus spp. over Various Temporal Scales at Three California Marine Beaches

2011 ◽  
Vol 78 (4) ◽  
pp. 1237-1242 ◽  
Author(s):  
Reagan R. Converse ◽  
John F. Griffith ◽  
Rachel T. Noble ◽  
Richard A. Haugland ◽  
Kenneth C. Schiff ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Membrane Filtration ◽  
Empirical Relationship ◽  
Time Of Day ◽  
Most Probable Number ◽  
Spatial And Temporal Variability ◽  
Culture Methods ◽  
Fecal Indicator ◽  
Probable Number ◽  
Content Type

ABSTRACTSeveral studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source.Enterococcusspp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [P< 0.002]) than in the afternoon (0.18 < ρ < 0.45 [P< 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [P< 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [P< 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach.

scholarly journals Spatial and Temporal Variation in Enterococcal Abundance and Its Relationship to the Microbial Community in Hawaii Beach Sand and Water

2013 ◽  
Vol 79 (12) ◽  
pp. 3601-3609 ◽  
Author(s):  
Henglin Cui ◽  
Kun Yang ◽  
Eulyn Pagaling ◽  
Tao Yan
Keyword(s):  
Microbial Community ◽  
Temporal Variation ◽  
Beach Sand ◽  
Spatial And Temporal Variation ◽  
Rrna Gene ◽  
Fecal Indicator ◽  
Bacterial Populations ◽  
Mass Unit ◽  
Content Type ◽  
Beach Water

ABSTRACTRecent studies have reported high levels of fecal indicator enterococci in marine beach sand. This study aimed to determine the spatial and temporal variation of enterococcal abundance and to evaluate its relationships with microbial community parameters in Hawaii beach sand and water. Sampling at 23 beaches on the Island of Oahu detected higher levels of enterococci in beach foreshore sand than in beach water on a mass unit basis. Subsequent 8-week consecutive samplings at two selected beaches (Waialae and Kualoa) consistently detected significantly higher levels of enterococci in backshore sand than in foreshore/nearshore sand and beach water. Comparison between the abundance of enterococci and the microbial communities showed that enterococci correlated significantly with totalVibrioin all beach zones but less significantly with total bacterial density andEscherichia coli. Samples from the different zones of Waialae beach were sequenced by 16S rRNA gene pyrosequencing to determine the microbial community structure and diversity. The backshore sand had a significantly more diverse community and contained different major bacterial populations than the other beach zones, which corresponded to the spatial distribution pattern of enterococcal abundance. Taken together, multiple lines of evidence support the possibility of enterococci as autochthonous members of the microbial community in Hawaii beach sand.

scholarly journals Differential Decay of Enterococci and Escherichia coli Originating from Two Fecal Pollution Sources

2013 ◽  
Vol 79 (7) ◽  
pp. 2488-2492 ◽  
Author(s):  
Asja Korajkic ◽  
Brian R. McMinn ◽  
Valerie J. Harwood ◽  
Orin C. Shanks ◽  
G. Shay Fout ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fecal Indicator Bacteria ◽  
Fecal Pollution ◽  
Pollution Sources ◽  
Indicator Bacteria ◽  
Fecal Indicator ◽  
Content Type ◽  
Marine Habitats ◽  
Aquatic Mesocosms

ABSTRACTUsingin situsubtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater andEscherichia coliin marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

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