Detection of Fecal Bacteria and Source Tracking Identifiers in Environmental Waters Using rRNA-Based RT-qPCR and rDNA-Based qPCR Assays

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (<i>n</i> = 14), surface water (<i>n</i> = 30), and treated source water (<i>n</i> = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for <i>E. coli</i> and <i>Campylobacter</i> spp. in surface waters, and for human-specific <i>Bacteroidales</i>, <i>E. coli</i>, and <i>Enterococcus</i> spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (<i>p</i> < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of <i>E. coli</i>, intestinal enterococci, and thermotolerant <i>Campylobacter</i> spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments

Detection of Fecal Bacteria and Source Tracking Identifiers in Environmental Waters Using rRNA-Based RT-qPCR and rDNA-Based qPCR Assays

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (<i>n</i> = 14), surface water (<i>n</i> = 30), and treated source water (<i>n</i> = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for <i>E. coli</i> and <i>Campylobacter</i> spp. in surface waters, and for human-specific <i>Bacteroidales</i>, <i>E. coli</i>, and <i>Enterococcus</i> spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (<i>p</i> < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of <i>E. coli</i>, intestinal enterococci, and thermotolerant <i>Campylobacter</i> spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments

Detection of Fecal Bacteria and Source Tracking Identifiers in Environmental Waters Using rRNA-Based RT-qPCR and rDNA-Based qPCR Assays

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (<i>n</i> = 14), surface water (<i>n</i> = 30), and treated source water (<i>n</i> = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for <i>E. coli</i> and <i>Campylobacter</i> spp. in surface waters, and for human-specific <i>Bacteroidales</i>, <i>E. coli</i>, and <i>Enterococcus</i> spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (<i>p</i> < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of <i>E. coli</i>, intestinal enterococci, and thermotolerant <i>Campylobacter</i> spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments

Categorized distance and proportion of cases within those groups in a waterborne outbreak in Vuorela, July 2012.

<p>Categorized distance and proportion of cases within those groups in a waterborne outbreak in Vuorela, July 2012.</p

Epidemic curve of a waterborne outbreak in Vuorela, July 2012 based on the reported onset date of illness of the cases, and <i>E. coli</i> bacteria counts and chlorine levels in the point 7 (See Fig. 1) of the water distribution network.

<p>Epidemic curve of a waterborne outbreak in Vuorela, July 2012 based on the reported onset date of illness of the cases, and <i>E. coli</i> bacteria counts and chlorine levels in the point 7 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104713#pone-0104713-g001" target="_blank">Fig. 1</a>) of the water distribution network.</p

The Univariate and multivariate results for individual risk factors and the generalized additive model risk ratios with the spatial term of a waterborne outbreak in Vuorela, July 2012.

<p>The Univariate and multivariate results for individual risk factors and the generalized additive model risk ratios with the spatial term of a waterborne outbreak in Vuorela, July 2012.</p

Microbiological results of faecal samples from symptomatic patients of a waterborne outbreak in Vuorela, July 2012.

<p>Methods used were cultivation¹, PCR/RT-PCR² (polymerase chain reaction/reverse transcripition-PCR), EM³ (electron microscopy) and/or EIA⁴ (Enzyme Immunoassay). ⁵<i>Campylobacter jejuni</i> and ⁶EHEC O157:H7 (from one sample) and ⁷sapovirus GII.P3 were isolated from the samples.</p

Phylogenetic relationships among OTUs (•) of the genus <i>Arcobacter</i> in a waterborne outbreak in Vuorela, July 2012.

<p>The tree was inferred from a maximum likelihood analysis of aligned 16S rRNA gene sequence (≈255 bp) and nodes with a bootstrap value ≥50% of 1 000 replicates are identified. <i>Sulfurospirillum deleyianum</i> (NR_074378) and <i>Campylobacter fetus</i> (L04314) were used as outgroup. Number in bracket represents the total amount of DNA/RNA reads identified in samples from A) the upper storage reservoir before cleaning, B) tap water during contamination and C) the upper storage reservoir after cleaning. *<i>A. butzleri</i>, <i>A. cryaerophilus</i> and <i>A. skirrowii</i> have been associated with gastrointestinal diseases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104713#pone.0104713-Collado1" target="_blank">[76]</a>.</p