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 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 Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces

2008 ◽  
Vol 74 (13) ◽  
pp. 3969-3976 ◽  
Author(s):  
Jingrang Lu ◽  
Jorge W. Santo Domingo ◽  
Regina Lamendella ◽  
Thomas Edge ◽  
Stephen Hill
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Diversity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Fecal Dna ◽  
Environmental Waters ◽  
Potential Risks ◽  
Species Specific ◽  
Gut Microbial Community

ABSTRACT In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium, a gram-positive, catalase-negative bacterium. To determine the occurrence of C. marimammalium in waterfowl, species-specific 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird (n = 13) and mammal (n = 26) species. The results showed that both assays were specific to gull fecal DNA and that C. marimammalium was present in gull fecal samples collected from the five locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C. marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters.

scholarly journals Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

scholarly journals Freshwater Suspended Sediments and Sewage Are Reservoirs for Enterotoxin-Positive Clostridium perfringens

2010 ◽  
Vol 76 (16) ◽  
pp. 5556-5562 ◽  
Author(s):  
Sabrina R. Mueller-Spitz ◽  
Lisa B. Stewart ◽  
J. Val Klump ◽  
Sandra L. McLellan
Keyword(s):  
Clostridium Perfringens ◽  
Lake Michigan ◽  
Suspended Sediments ◽  
Fecal Pollution ◽  
Gene Arrangement ◽  
Toxin Gene ◽  
Fecal Indicator ◽  
Specific Pcr ◽  
Enterotoxin Genes ◽  
Species Specific

ABSTRACT The release of fecal pollution into surface waters may create environmental reservoirs of feces-derived microorganisms, including pathogens. Clostridium perfringens is a commonly used fecal indicator that represents a human pathogen. The pathogenicity of this bacterium is associated with its expression of multiple toxins; however, the prevalence of C. perfringens with various toxin genes in aquatic environments is not well characterized. In this study, C. perfringens spores were used to measure the distribution of fecal pollution associated with suspended sediments in the nearshore waters of Lake Michigan. Particle-associated C. perfringens levels were greatest adjacent to the Milwaukee harbor and diminished in the nearshore waters. Species-specific PCR and toxin gene profiles identified 174 isolates collected from the suspended sediments, surface water, and sewage influent as C. perfringens type A. Regardless of the isolation source, the beta2 and enterotoxin genes were common among isolates. The suspended sediments yielded the highest frequency of cpe-carrying C. perfringens (61%) compared to sewage (38%). Gene arrangement of enterotoxin was investigated using PCR to target known insertion sequences associated with this gene. Amplification products were detected in only 9 of 90 strains, which suggests there is greater variability in cpe gene arrangement than previously described. This work presents evidence that freshwater suspended sediments and sewage influent are reservoirs for potentially pathogenic cpe-carrying C. perfringens spores.

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 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 Distributions of Fecal Markers in Wastewater from Different Climatic Zones for Human Fecal Pollution Tracking in Australian Surface Waters

2015 ◽  
Vol 82 (4) ◽  
pp. 1316-1323 ◽  
Author(s):  
W. Ahmed ◽  
J. P. S. Sidhu ◽  
K. Smith ◽  
D. J. Beale ◽  
P. Gyawali ◽  
...  
Keyword(s):  
Municipal Wastewater ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Environmental Waters ◽  
Fecal Indicator ◽  
Climatic Zones ◽  
Content Type ◽  
Gene Copies ◽  
Viral Marker ◽  
Fecal Markers

ABSTRACTRecreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB)Escherichia coliandEnterococcusspp., HFMsBacteroidesHF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia.E. colimean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 106gene copies per ml), followed by those of HF183 (8.0 × 105gene copies per ml) andEnterococcusspp. (3.6 × 105gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated thatBacteroidesHF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters.

scholarly journals Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

2006 ◽  
Vol 72 (12) ◽  
pp. 7567-7574 ◽  
Author(s):  
Shannon M. McQuaig ◽  
Troy M. Scott ◽  
Valerie J. Harwood ◽  
Samuel R. Farrah ◽  
Jerzy O. Lukasik
Keyword(s):  
Water Samples ◽  
Environmental Water ◽  
Fecal Pollution ◽  
Fecal Coliforms ◽  
Human Polyomavirus ◽  
Environmental Water Samples ◽  
Human Populations ◽  
Rrna Gene ◽  
Recreational Water ◽  
Environmental Waters

ABSTRACT Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”

scholarly journals Cross-Comparison of Human Wastewater-Associated Molecular Markers in Relation to Fecal Indicator Bacteria and Enteric Viruses in Recreational Beach Waters

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
B. Hughes ◽  
D. J. Beale ◽  
P. G. Dennis ◽  
S. Cook ◽  
W. Ahmed
Keyword(s):  
Water Samples ◽  
Enteric Viruses ◽  
Human Adenovirus ◽  
Fecal Pollution ◽  
Treated Wastewater ◽  
Recreational Water ◽  
Environmental Waters ◽  
Fecal Indicator ◽  
Content Type ◽  
Beach Water

ABSTRACT Detection of human wastewater contamination in recreational waters is of critical importance to regulators due to the risks posed to public health. To identify such risks, human wastewater-associated microbial source tracking (MST) markers have been developed. At present, however, a greater understanding of the suitability of these markers for the detection of diluted human wastewater in environmental waters is necessary to predict risk. Here, we compared the process limit of detection (PLOD) and process limit of quantification (PLOQ) of six human wastewater-associated MST markers (Bacteroides HF183 [HF183], Escherichia coli H8 [EC H8], Methanobrevibacter smithii nifH, human adenovirus [HAdV], human polyomavirus [HPyV], and pepper mild mottle virus [PMMoV]) in relation to a fecal indicator bacterium (FIB), Enterococcus sp. 23S rRNA (ENT 23S), and three enteric viruses (human adenovirus serotypes 40/41 [HAdV 40/41], human norovirus [HNoV], and human enterovirus [EV]) in beach water samples seeded with raw and secondary-treated wastewater. Among the six MST markers tested, HF183 was the most sensitive measure of human fecal pollution and was quantifiable up to dilutions of 10−6 and 10−4 for beach water samples seeded with raw and secondary-treated wastewater, respectively. Other markers and enteric viruses were detected at various dilutions (10−1 to 10−5). These MST markers, FIB, and enteric viruses were then quantified in beach water (n = 12) and sand samples (n = 12) from South East Queensland (SEQ), Australia, to estimate the levels of human fecal pollution. Of the 12 sites examined, beach water and sand samples from several sites had quantifiable concentrations of HF183 and PMMoV markers. Overall, our results indicate that while HF183 is the most sensitive measure of human fecal pollution, it should be used in conjunction with a conferring viral marker to avoid overestimating the risk of gastrointestinal illness. IMPORTANCE MST is an effective tool to help utilities and regulators improve recreational water quality around the globe. Human fecal pollution poses significant public health risks compared to animal fecal pollution. Several human wastewater-associated markers have been developed and used for MST field studies. However, a head-to-head comparison in terms of their performance to detect diluted human fecal pollution in recreational water is lacking. In this study, we cross-compared the performance of six human wastewater-associated markers in relation to FIB and enteric viruses in beach water samples seeded with raw and secondary-treated wastewater. The results of this study will provide guidance to regulators and utilities on the appropriate application of MST markers for tracking the sources of human fecal pollution in environmental waters and confer human health risks.

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