Potential Human Pathogenic Bacteria in a Mixed Urban Watershed as Revealed by Pyrosequencing (original) (raw)
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Microbiome of Bacterially Impaired Watersheds: Distribution of Potential Bacterial Pathogens
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Bacterial impairment of freshwater systems is a commonly studied global problem. However, studies on the relative distribution of bacterial pathogens in different impaired aquatic systems have been limited. Frequently, impaired freshwater systems are classified by the presence of fecal indicator bacteria (FIB) and the identification of sources of fecal contamination through microbial source tracking. In this study, we assessed the relative abundance of DNA sequences related to potential human bacterial pathogens along with human fecal indicator bacteria in three impaired watersheds. These watersheds consistently showed a high abundance of FIB for the past several years. Using Illumina paired-end DNA sequencing of 16S rRNA gene amplicons, we observed variation in the relative distribution of DNA sequences related to Legionellaceae, Enterobacteriaceae and Bacteroidaceae families across different sites. We identified potential hotspots sites in these impaired water systems, which showe...
Journal of Water and Health
Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has been under the longest active boil water advisory in Canada due to FIB contamination in groundwater wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also...
Applied and environmental microbiology, 2015
The 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, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR a...
Applied and Environmental Microbiology, 2006
Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds.
The field of molecular fecal source tracking in the water environment has developed rapidly since the first PCR assays for general and host-‐specific Bacteroides 16s rRNA markers were published. Numerous host-‐specific molecular markers and PCR assays have been developed, adding greater specificity, sensitivity and quantitative methods to the array of options. The public demand for readying methods for transfer to the commercial lab, so that they may be used to generate data for public utilities, citizen action groups and regulatory agencies, has fueled the development of an entire new research community. These methods, however plentiful, have not found community agreement and there is no consensus concerning the appropriate implementation of molecular fecal source tracking in the field. Some issues plaguing the implementation include imperfect marker specificity, environmental variability, DNA extraction variability, PCR inhibition and high cost of molecular analysis. This thesis...
Fems Microbiology Ecology, 2007
We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28–54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3–5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19–24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.
Effective and sensitive monitoring of human pathogenic bacteria in municipal wastewater treatment is important not only for managing public health risk related to treated wastewater reuse, but also for ensuring proper functioning of the treatment plant. In this study, three different 16S rRNA gene molecular analysis methodologies were employed to screen bacterial pathogens in samples collected at three different stages of an activated sludge plant. Overall bacterial diversity was analyzed using next generation sequencing (NGS) on the Illumina MiSeq platform, as well as PCR-DGGE followed by band sequencing. In addition, a microdiversity analysis was conducted using PCR-DGGE, targeting Escherichia coli. Bioinformatics analysis was performed using QIIME protocol by clustering sequences against the Human Pathogenic Bacteria Database. NGS data were also clustered against the Greengenes database for a genera-level diversity analysis. NGS proved to be the most effective approach screening the sequences of 21 potential human bacterial pathogens, while the E. coli microdiversity analysis yielded one (O157:H7 str. EDL933) out of the two E. coli strains picked up by NGS. Overall diversity using PCR-DGGE did not yield any pathogenic sequence matches even though a number of sequences matched the NGS results. Overall, sequences of Gramnegative pathogens decreased in relative abundance along the treatment train while those of Gram-positive pathogens increased.
FEMS Microbiology Reviews, 2014
Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms. Human sewage pollution is among the greatest concerns for human health due to (1) the known risk of exposure to human waste and (2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology.