16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

Revetta, Randy P.; Matlib, Robin S.; Domingo, Jorge W. Santo

doi:10.1007/s00284-011-9938-9

Cited by 36 publications

(37 citation statements)

References 24 publications

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“…Here, we applied rRNA based RTqPCR assays for the identification and quantification of fecal bacteria and human-specific markers in an urban watershed impacted by fecal contamination. The greater sensitivity of rRNAbased assays over the rRNA gene-based detection obtained in our results is also consistent with previous studies targeting RNA and DNA based on molecular markers for enumeration of bacteria in drinking water (46,47), in coastal and environmental waters (30,48), including waters impacted with fecal pollution (27), and in soil and sediments (26,49,50). The signals of human-specific Bacteroidales, E. coli, and Enterococcus spp.…”

Section: Discussionsupporting

confidence: 80%

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

Kapoor

Pitkänen

Ryu

et al. 2015

Appl Environ Microbiol

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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 assay counterpart. On average, two human-specific Bacteroidales markers 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-specific Bacteroidales markers 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. Sewage overflows and stormwater runoff introduce high levels of fecal bacteria into surface waters and are considered the primary cause of water quality impairments in urban watersheds, particularly those affected by combined sewer overflows (CSOs) (1, 2). Sewage contamination of surface waters poses a serious risk to human and environmental health via waterborne disease outbreaks (3-5), deterioration of recreational and drinking water quality (6, 7), and degradation of aquatic ecology (8, 9). Hence, identifying the primary source(s) of fecal contamination is imperative to enable best management practices for mitigating pollution and public health risks.Microbial source tracking (MST) methods targeting fecal bacteria have recently been used to identify the sources of fecal contamination impacting water systems (10, 11). Many of these MST methods are based on quantitative PCR (qPCR) assays targeting the bacterial rRNA genes present within water DNA extracts (12, 13). However, the value of DNA-based monitoring in microbial ecology studies is limited by the possibility of DNA being associated with dead cells or the extent to which "naked DNA" may survive in the environment once bacteria are lysed (14-16). These facts pose a significant challenge to the environmental fate and transport of fecal bacteria which are often assess...

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Section: Discussionsupporting

confidence: 80%

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

Kapoor

Pitkänen

Ryu

et al. 2015

Appl Environ Microbiol

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“…The presence of sequences exclusively found in one of the two nucleic acid pools suggests that using both nucleic acids provides a more complete overview of the microbial diversity present in a given environment since on average, only 20 % of the OTUs (18-22 %) were shared per DO zone and per season. As previously suggested [81], sequences found only in the DNA pool might be related to metabolically less active, yet abundant populations, whereas sequences detected only in RNA are associated with active members of the community but may be present in relatively low numbers. The type of populations associated with shared and unshared sequences could be the result of differences in their respective overall abundance and/or a reflection on the overall metabolic status of a population within a given OTU.…”

Section: Discussionmentioning

confidence: 52%

Vertical Distribution of Functional Potential and Active Microbial Communities in Meromictic Lake Kivu

et al. 2015

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The microbial community composition in meromictic Lake Kivu, with one of the largest CH 4 reservoirs, was studied using 16S rDNA and ribosomal RNA (rRNA) pyrosequencing during the dry and rainy seasons. Highly abundant taxa were shared in a high percentage between bulk (DNA-based) and active (RNA-based) bacterial communities, whereas a high proportion of rare species was detected only in either an active or bulk community, indicating the existence of a potentially active rare biosphere and the possible underestimation of diversity detected when using only one nucleic acid pool. Most taxa identified as generalists were abundant, and those identified as specialists were more likely to be rare in the bulk community. The overall number of environmental parameters that could explain the variation was higher for abundant taxa in comparison to rare taxa. Clustering analysis based on operational taxonomic units (OTUs at 0.03 cutoff) level revealed significant and systematic microbial community composition shifts with depth. In the oxic zone, Actinobacteria were found highly dominant in the bulk community but not in the metabolically active community. In the oxic-anoxic transition zone, highly abundant potentially active Nitrospira and Methylococcales were observed. The cooccurrence of potentially active sulfur-oxidizing and sulfatereducing bacteria in the anoxic zone may suggest the presence of an active yet cryptic sulfur cycle.

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“…to be sloughed from the biofilms should be further confirmed. Other studies have demonstrated that not only are Cyanobacteria present in chlorinated DWDS but some are also potentially active members of the DWDS microbial community (17,31), suggesting that they could survive from different physical and chemical treatments and enter the DWDS (13). We think the use of cultivationbased methods together with metagenomics approaches should be pursued to further understand the ecological roles of predominant populations in DWDS.…”

Section: Discussionmentioning

confidence: 99%

Microbial Community Dynamics of an Urban Drinking Water Distribution System Subjected to Phases of Chloramination and Chlorination Treatments

Hwang

Ling

Andersen

et al. 2012

Appl Environ Microbiol

128

103

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Water utilities in parts of the U.S. control microbial regrowth in drinking water distribution systems (DWDS) by alternating postdisinfection methods between chlorination and chloramination. To examine how this strategy influences drinking water microbial communities, an urban DWDS (population Х 40,000) with groundwater as the source water was studied for approximately 2 years. Water samples were collected at five locations in the network at different seasons and analyzed for their chemical and physical characteristics and for their microbial community composition and structure by examining the 16S rRNA gene via terminal restriction fragment length polymorphism and DNA pyrosequencing technology. Nonmetric multidimension scaling and canonical correspondence analysis of microbial community profiles could explain >57% of the variation. Clustering of samples based on disinfection types (free chlorine versus combined chlorine) and sampling time was observed to correlate to the shifts in microbial communities. Sampling location and water age (<21.2 h) had no apparent effects on the microbial compositions of samples from most time points. Microbial community analysis revealed that among major core populations, Cyanobacteria, Methylobacteriaceae, Sphingomonadaceae, and Xanthomonadaceae were more abundant in chlorinated water, and Methylophilaceae, Methylococcaceae, and Pseudomonadaceae were more abundant in chloraminated water. No correlation was observed with minor populations that were detected frequently (<0.1% of total pyrosequences), which were likely present in source water and survived through the treatment process. Transient microbial populations including Flavobacteriaceae and Clostridiaceae were also observed. Overall, reversible shifts in microbial communities were especially pronounced with chloramination, suggesting stronger selection of microbial populations from chloramines than chlorine.

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16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

Cited by 36 publications

References 24 publications

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

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

Vertical Distribution of Functional Potential and Active Microbial Communities in Meromictic Lake Kivu

Microbial Community Dynamics of an Urban Drinking Water Distribution System Subjected to Phases of Chloramination and Chlorination Treatments

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