Distribution of Genetic Marker Concentrations for Fecal Indicator Bacteria in Sewage and Animal Feces

Kelty, Catherine A.; Varma, Manju; Sivaganesan, Mano; Haugland, Richard A.; Shanks, Orin C.

doi:10.1128/aem.07819-11

Cited by 35 publications

(35 citation statements)

References 44 publications

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“…Another recent report found low densities of Bacteroidales in soils (2.5 log gene copies g Ϫ1 ) and chlorinated tap water (1.4 log gene copies liter Ϫ1 ) (33). Relatively few reports have presented evidence of Bacteroidales densities in chicken and turkey feces (6,34) and in poultry litter (33,35). Additionally, we were unable to find peer-reviewed reports regarding the fate of Bacteroidales originating from poultry feces and deposited in poultry litter.…”

contrasting

confidence: 40%

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Weidhaas

Mantha

Hair

et al. 2015

Appl Environ Microbiol

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bWater quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have 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 of Bacteroides in 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 of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml ؊1 and 2 log gene copies g litter ؊1 under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.

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contrasting

confidence: 40%

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Weidhaas

Mantha

Hair

et al. 2015

Appl Environ Microbiol

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“…This approach has some advantages such as avoiding the need to measure and correct for DNA extraction efficiencies and error introduced by sample variability (solid or liquid phases). The standardization process may also relieve PCR inhibition [77]. The mean concentrations of HF183 and HuBac in human feces were approximately two orders of magnitude higher than BacHum-UCD and BacH markers.…”

Section: Human Feces and Wastewatermentioning

confidence: 95%

Current Status of Marker Genes of Bacteroides and Related Taxa for Identifying Sewage Pollution in Environmental Waters

Ahmed

Hughes

Harwood

2016

Water

112

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Abstract:Microbial source tracking (MST) endeavors to determine sources of fecal pollution in environmental waters by capitalizing on the association of certain microorganisms with the gastrointestinal tract and feces of specific animal groups. Several decades of research have shown that bacteria belonging to the gut-associated order Bacteroidales, and particularly the genus Bacteroides, tend to co-evolve with the host, and are, therefore, particularly suitable candidates for MST applications. This review summarizes the current research on MST methods that employ genes belonging to Bacteroidales/Bacteroides as tracers or "markers" of sewage pollution, including known advantages and deficiencies of the many polymerase chain reaction (PCR)-based methods that have been published since 2000. Host specificity is a paramount criterion for confidence that detection of a marker is a true indicator of the target host. Host sensitivity, or the prevalence of the marker in feces/waste from the target host, is necessary for confidence that absence of the marker is indicative of the absence of the pollution source. Each of these parameters can vary widely depending on the type of waste assessed and the geographic location. Differential decay characteristics of bacterial targets and their associated DNA contribute to challenges in interpreting MST results in the context of human health risks. The HF183 marker, derived from the 16S rRNA gene of Bacteroides dorei and closely related taxa, has been used for almost two decades in MST studies, and is well characterized regarding host sensitivity and specificity, and in prevalence and concentration in sewage in many countries. Other markers such as HumM2 and HumM3 show promise, but require further performance testing to demonstrate their widespread utility. An important limitation of the one-marker-one-assay approach commonly used for MST is that given the complexities of microbial persistence in environmental waters, and the methodological challenges of quantitative PCR (qPCR) in such samples, the absence of a given marker does not ensure the absence of fecal pollution in the source water. Approaches under development, such as microarray and community analysis, have the potential to improve MST practices, thereby increasing our ability to protect human and ecosystem health.

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“…All DNA extractions were performed with the DNA-EZ RW02 kit (GeneRite LLC, North Brunswick, NJ) according to the manufacturer's instructions, as previously described (34). For all filters including extraction blanks, 800 l of 0.2 g/ml salmon testes DNA diluted in AE buffer (Qiagen, Valencia, CA) was spiked into each bead milling tube prior to extraction (29).…”

Section: Methodsmentioning

confidence: 99%

“…The range of standard concentrations where at least two or more of the three replicates pass (VIC C q Յ interference threshold) for each standard dilution indicated the respective instrument run-specific IAC ROQ. The competition thresholds were defined as the calibration model FAM C q value that intersects the upper bound of a respective instrument run-specific IAC ROQ (34). Any filter DNA extract exhibiting amplification interference (determined from the IAC assay VIC C q measurements), where the filter mean FAM C q value from the native sequence target assay (calculated from the filter DNA extract triplicate FAM C q measurements) was greater than the respective competition threshold, indicated inhibition and was discarded from the study.…”

Section: Methodsmentioning

confidence: 99%

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

Shanks

Kelty

Oshiro

et al. 2016

Appl Environ Microbiol

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There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (notemplate and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark. Fecal pollution remains a significant challenge for ambient water quality managers worldwide. In the United States alone, it is estimated that 39.2% of all rivers, lakes, and streams are unsafe for recreational use, with fecal pathogens as the number one cause of impairment (1). General fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, shed by nearly all warm-blooded animals, are routinely used to assess water quality. However, general FIB cannot discriminate between different animal groups, making it challenging to pinpoint the origin of fecal pollution. Researchers and managers alike recognize the advantages of animal source information to help solve long-standing ambient water quality problems. As a result, a number of fecal source identification technologies have been developed (2-9). These methods have been employed to address challenges such as the identification ...

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Distribution of Genetic Marker Concentrations for Fecal Indicator Bacteria in Sewage and Animal Feces

Cited by 35 publications

References 44 publications

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Current Status of Marker Genes of Bacteroides and Related Taxa for Identifying Sewage Pollution in Environmental Waters

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

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