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 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 LA35 Poultry Fecal Marker Persistence Is Correlated with That of Indicators and Pathogens in Environmental Waters

2015 ◽  
Vol 81 (14) ◽  
pp. 4616-4625 ◽  
Author(s):  
Bina Nayak ◽  
Jennifer Weidhaas ◽  
Valerie J. Harwood
Keyword(s):  
Water Quality ◽  
Health Risk ◽  
Water Column ◽  
Poultry Litter ◽  
Land Application ◽  
Gene Marker ◽  
Rrna Gene ◽  
Content Type ◽  
The Impact ◽  
Over Time

ABSTRACTDisposal of fecally contaminated poultry litter by land application can deliver pathogens and fecal indicator bacteria (FIB) into receiving waters via runoff. While water quality is regulated by FIB enumeration, FIB testing provides inadequate information about contamination source and health risk. This microbial source tracking (MST) study compared the persistence of theBrevibacteriumsp. strain LA35 16S rRNA gene (marker) for poultry litter with that of pathogens and FIB under outdoor, environmentally relevant conditions in freshwater, marine water, and sediments over 7 days.Salmonella enterica,Campylobacter jejuni,Campylobacter coli,Bacteroidales, and LA35 were enumerated by quantitative PCR (qPCR), andEnterococcusspp. andE. coliwere quantified by culture and qPCR. Unlike the other bacteria,C. jejuniwas not detectable after 48 h. Bacterial levels in the water column consistently declined over time and were highly correlated among species. Survival in sediments ranged from a slow decrease over time to growth, particularly in marine microcosms and forBacteroidales. S. entericaalso grew in marine sediments. Linear decay rates in water (k) ranged from −0.17 day−1for LA35 to −3.12 day−1forC. coli. LA35 levels correlated well with those of other bacteria in the water column but not in sediments. These observations suggest that, particularly in the water column, the fate of LA35 in aquatic environments is similar to that of FIB,C. coli, andSalmonella, supporting the hypothesis that the LA35 marker gene can be a useful tool for evaluating the impact of poultry litter on water quality and human health risk.

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 Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

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 Identification of a Brevibacterium Marker Gene Specific to Poultry Litter and Development of a Quantitative PCR Assay

2009 ◽  
Author(s):  
Jennifer L. Weidhaas ◽  
Tamzen W. Macbeth ◽  
Roger L. Olsen ◽  
Michael J. Sadowsky ◽  
Daniel Norat ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Marker Gene ◽  
Poultry Litter ◽  
Pcr Assay ◽  
Quantitative Pcr Assay

scholarly journals Bifidobacteria in Feces and Environmental Waters

2007 ◽  
Vol 74 (3) ◽  
pp. 575-584 ◽  
Author(s):  
Regina Lamendella ◽  
Jorge W. Santo Domingo ◽  
Catherine Kelty ◽  
Daniel B. Oerther
Keyword(s):  
Natural Waters ◽  
Wastewater Treatment Plants ◽  
Anaerobic Bacteria ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Environmental Waters ◽  
Fecal Indicator ◽  
Bifidobacterium Adolescentis ◽  
Indicator Group ◽  
Species Specific

ABSTRACT Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.

scholarly journals Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

2007 ◽  
Vol 74 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Orin C. Shanks ◽  
Emina Atikovic ◽  
A. Denene Blackwood ◽  
Jingrang Lu ◽  
Rachel T. Noble ◽  
...  
Keyword(s):  
Genetic Marker ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Broad Distribution ◽  
Assay Performance ◽  
Target Dna ◽  
Bovine Feces

ABSTRACTAccurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing totalBacteroidetes,Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient 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.

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