scholarly journals Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

2016 ◽  
Vol 82 (5) ◽  
pp. 1625-1635 ◽  
Author(s):  
Xiang Li ◽  
Valerie J. Harwood ◽  
Bina Nayak ◽  
Jennifer L. Weidhaas
Keyword(s):  
Legionella Pneumophila ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Marine Water ◽  
Treated Wastewater ◽  
Human Polyomavirus ◽  
Source Tracking ◽  
Marker Genes ◽  
Content Type ◽  
Gene Copies

ABSTRACTPathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% forEscherichia coliand human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such asLegionella pneumophila,Shigella flexneri, andCampylobacter fetuswere detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.

scholarly journals Evaluation of Bovine Feces-Associated Microbial Source Tracking Markers and Their Correlations with Fecal Indicators and Zoonotic Pathogens in a Brisbane, Australia, Reservoir

2013 ◽  
Vol 79 (8) ◽  
pp. 2682-2691 ◽  
Author(s):  
W. Ahmed ◽  
T. Sritharan ◽  
A. Palmer ◽  
J. P. S. Sidhu ◽  
S. Toze
Keyword(s):  
Host Specificity ◽  
Water Samples ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Decay Rates ◽  
Source Tracking ◽  
Content Type ◽  
Zoonotic Pathogens ◽  
Maximum Value ◽  
Bovine Feces

ABSTRACTThis study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral [bovine adenovirus (B-AVs)] microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB),Campylobacterspp.,Escherichia coliO157, andSalmonellaspp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for theCampylobacter16S rRNA andE. coliO157rfbEgenes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens.

scholarly journals Automated Sampling Procedures Supported by High Persistence of Bacterial Fecal Indicators and Bacteroidetes Genetic Microbial Source Tracking Markers in Municipal Wastewater during Short-Term Storage at 5°C

2015 ◽  
Vol 81 (15) ◽  
pp. 5134-5143 ◽  
Author(s):  
R. E. Mayer ◽  
J. Vierheilig ◽  
L. Egle ◽  
G. H. Reischer ◽  
E. Saracevic ◽  
...  
Keyword(s):  
Municipal Wastewater ◽  
Storage Conditions ◽  
Microbial Source Tracking ◽  
Basic Knowledge ◽  
Treated Wastewater ◽  
Source Tracking ◽  
Fecal Indicators ◽  
Content Type ◽  
Automated Sampling ◽  
Sampling Procedures

ABSTRACTBecause of high diurnal water quality fluctuations in raw municipal wastewater, the use of proportional autosampling over a period of 24 h at municipal wastewater treatment plants (WWTPs) to evaluate carbon, nitrogen, and phosphorus removal has become a standard in many countries. Microbial removal or load estimation at municipal WWTPs, however, is still based on manually recovered grab samples. The goal of this study was to establish basic knowledge regarding the persistence of standard bacterial fecal indicators andBacteroidetesgenetic microbial source tracking markers in municipal wastewater in order to evaluate their suitability for automated sampling, as the potential lack of persistence is the main argument against such procedures. Raw and secondary treated wastewater of municipal origin from representative and well-characterized biological WWTPs without disinfection (organic carbon and nutrient removal) was investigated in microcosm experiments at 5 and 21°C with a total storage time of 32 h (including a 24-h autosampling component and an 8-h postsampling phase). VegetativeEscherichia coliand enterococci, as well asClostridium perfringensspores, were selected as indicators for cultivation-based standard enumeration. Molecular analysis focused on total (AllBac) and human-associated geneticBacteroidetes(BacHum-UCD, HF183 TaqMan) markers by using quantitative PCR, as well as 16S rRNA gene-based next-generation sequencing. The microbial parameters showed high persistence in both raw and treated wastewater at 5°C under the storage conditions used. Surprisingly, and in contrast to results obtained with treated wastewater, persistence of the microbial markers in raw wastewater was also high at 21°C. On the basis of our results, 24-h autosampling procedures with 5°C storage conditions can be recommended for the investigation of fecal indicators orBacteroidetesgenetic markers at municipal WWTPs. Such autosampling procedures will contribute to better understanding and monitoring of municipal WWTPs as sources of fecal pollution in water resources.

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 Association of Fecal Indicator Bacteria with Human Viruses and Microbial Source Tracking Markers at Coastal Beaches Impacted by Nonpoint Source Pollution

2012 ◽  
Vol 78 (18) ◽  
pp. 6423-6432 ◽  
Author(s):  
Shannon McQuaig ◽  
John Griffith ◽  
Valerie J. Harwood
Keyword(s):  
Water Quality ◽  
Fecal Indicator Bacteria ◽  
Human Health Risk ◽  
Microbial Source Tracking ◽  
Indicator Bacteria ◽  
Fecal Coliforms ◽  
Human Polyomavirus ◽  
Source Tracking ◽  
Fecal Indicator ◽  
Content Type

ABSTRACTWater quality was assessed at two marine beaches in California by measuring the concentrations of culturable fecal indicator bacteria (FIB) and by library-independent microbial source tracking (MST) methods targeting markers of human-associated microbes (human polyomavirus [HPyV] PCR and quantitative PCR,Methanobrevibacter smithiiPCR, andBacteroidessp. strain HF183 PCR) and a human pathogen (adenovirus by nested PCR). FIB levels periodically exceeded regulatory thresholds at Doheny and Avalon Beaches for enterococci (28.5% and 31.7% of samples, respectively) and fecal coliforms (20% and 5.8%, respectively). Adenoviruses were detected at four of five sites at Doheny Beach and were correlated with detection of HPyVs and humanBacteroidesHF183; however, adenoviruses were not detected at Avalon Beach. The most frequently detected human source marker at both beaches wasBacteroidesHF183, which was detected in 27% of samples. Correlations between FIBs and human markers were much more frequent at Doheny Beach than at Avalon Beach; e.g., adenovirus was correlated with HPyVs and HF183. Human sewage markers and adenoviruses were routinely detected in samples meeting FIB regulatory standards. The toolbox approach of FIB measurement coupled with analysis of several MST markers targeting human pathogens used here demonstrated that human sewage is at least partly responsible for the degradation of water quality, particularly at Doheny Beach, and resulted in a more definitive assessment of recreational water quality and human health risk than reliance on FIB concentrations alone could have provided.

scholarly journals Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

2014 ◽  
Vol 81 (1) ◽  
pp. 196-202 ◽  
Author(s):  
Jennifer Weidhaas ◽  
Sirisha Mantha ◽  
Elliott Hair ◽  
Bina Nayak ◽  
Valerie J. Harwood
Keyword(s):  
Poultry Litter ◽  
Water Quality Monitoring ◽  
Quality Monitoring ◽  
Source Tracking ◽  
Environmental Waters ◽  
Fecal Indicator ◽  
Content Type ◽  
Gene Copies ◽  
Laboratory Microcosm ◽  
Host Feces

ABSTRACTWater quality monitoring techniques that target microorganisms in the orderBacteroidalesare potential alternatives to conventional methods for detection of fecal indicator bacteria.Bacteroidalesand members of the genusBacteroideshave been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance ofBacteroidesin host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth ofBacteroidalesoriginating from poultry litter under oxic and anoxic environmental conditions.Bacteroidalesabundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml−1and 2 log gene copies g litter−1under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of theBacteroidalesorganisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related toBacteroides uniformis,B. ovatus, andB. vulgatus. These results suggest that MST studies using qPCR methods targetingBacteroidalesin watersheds that are affected by poultry litter should be interpreted cautiously. Growth ofBacteroidalesoriginating from poultry litter in environmental waters may occur whileBacteroidalesgrowth from other fecal sources declines, thus confounding the interpretation of MST results.

scholarly journals Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

2012 ◽  
Vol 78 (20) ◽  
pp. 7317-7326 ◽  
Author(s):  
Christopher Staley ◽  
Katrina V. Gordon ◽  
Mary E. Schoen ◽  
Valerie J. Harwood
Keyword(s):  
Risk Assessment ◽  
Quantitative Pcr ◽  
Microbial Source Tracking ◽  
Human Polyomavirus ◽  
Source Tracking ◽  
Specific Marker ◽  
Environmental Waters ◽  
Potential Health ◽  
Microbial Risk Assessment ◽  
Microbial Risk

ABSTRACTBefore new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associatedBacteroidessp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10−6in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10−4. Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context.

scholarly journals Identifying Host Sources of Fecal Pollution: Diversity of Escherichia coli in Confined Dairy and Swine Production Systems

2005 ◽  
Vol 71 (10) ◽  
pp. 5992-5998 ◽  
Author(s):  
Zexun Lu ◽  
David Lapen ◽  
Andrew Scott ◽  
Angela Dang ◽  
Edward Topp
Keyword(s):  
Escherichia Coli ◽  
Dairy Farm ◽  
Sampling Strategy ◽  
Farming Systems ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Source Tracking ◽  
E Coli ◽  
Repetitive Extragenic Palindromic Pcr ◽  
Repetitive Extragenic Palindromic

ABSTRACT Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

scholarly journals Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Shuchen Feng ◽  
Warish Ahmed ◽  
Sandra L. McLellan
Keyword(s):  
False Positive ◽  
Marker Gene ◽  
Fecal Pollution ◽  
Marker Genes ◽  
Indicator Organisms ◽  
Sequencing Data ◽  
Content Type ◽  
E Coli ◽  
Cross Reactions ◽  
Nonspecific Amplification

ABSTRACT Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene’s true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera Blautia or Bacteroides. For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human Bacteroides [HB]), and 94.0% (Lachno3). Select assays duplexed with either Escherichia coli or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples. IMPORTANCE Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below E. coli or Enterococcus levels and likely would not impact the assay’s ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.

scholarly journals Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

2013 ◽  
Vol 80 (2) ◽  
pp. 612-617 ◽  
Author(s):  
Kruti Ravaliya ◽  
Jennifer Gentry-Shields ◽  
Santos Garcia ◽  
Norma Heredia ◽  
Anna Fabiszewski de Aceituno ◽  
...  
Keyword(s):  
Fresh Produce ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rrna Gene ◽  
Specific Marker ◽  
Production Environment ◽  
Content Type ◽  
Food Borne

ABSTRACTIn recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based onBacteroidales16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universalBacteroidalesmarker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation betweenBacteroidalesand genericEscherichia coliacross all samples. This study provides evidence thatBacteroidalesmarkers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.

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