Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR

Chern, Eunice C.; Brenner, Kristen P.; Wymer, Larry; Haugland, Richard A.

doi:10.1007/s12403-009-0019-2

Cited by 56 publications

(53 citation statements)

References 45 publications

(47 reference statements)

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“…DNA standards for all qPCR assays were prepared from bacterial cultures as previously described (4,13,28). Cells were lysed in a bead mill for 60 s at maximum speed, and the debris was removed by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

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

Kelty

Varma

Sivaganesan

et al. 2012

Appl Environ Microbiol

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bVery little is known about the density and distribution of fecal indicator bacteria (FIB) genetic markers measured by quantitative real-time PCR (qPCR) in fecal pollution sources. Before qPCR-based FIB technologies can be applied to waste management and public health risk applications, it is vital to characterize the concentrations of these genetic markers in pollution sources (i.e., untreated wastewater and animal feces). We report the distribution of rRNA genetic markers for several general FIB groups, including Clostridium spp., Escherichia coli, enterococci, and Bacteroidales, as determined by qPCR on reference collections consisting of 54 primary influent sewage samples collected from treatment facilities across the United States and fecal samples representing 20 different animal species. Based on raw sewage sample collection data, individual FIB genetic markers exhibited a remarkable similarity in concentration estimates from locations across the United States ranging from Hawaii to Florida. However, there was no significant correlation between genetic markers for most FIB combinations (P > 0.05). In addition, large differences (up to 5 log 10 copies) in the abundance of FIB genetic markers were observed between animal species, emphasizing the importance of indicator microorganism selection and animal source contribution for future FIB applications.

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Section: Methodsmentioning

confidence: 99%

“…The four real-time PCR (qPCR) assays used in this study were Entero1, uidA, Cperf, and GenBac3 (4,7,20,28,35), for enterococci, E. coli, Clostridium spp., and Bacteroidales, respectively. The primer, TaqMan probe, and presumptive target organism(s) for each qPCR assay are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

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

Kelty

Varma

Sivaganesan

et al. 2012

Appl Environ Microbiol

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“…In order to select a Q-PCR assay and subsequently design RT-Q-PCR assays for E. coli with the highest levels of specificity, sensitivity, and efficiency, a literature review identified three frequently used functional genes for E. coli, uidA (24), tuf (25), and rodA (26) for comparison. The uidA gene (coding for ␤-D-glucuronidase) was chosen as the most frequently used target gene for E. coli (24,38,39,40,41).…”

Section: Test Panel Specificity and (Rt-)q-pcr Assaysmentioning

confidence: 99%

“…Water quality failures due to the survival or regrowth of E. coli within a WDS bring into question the suitability of E. coli as an indicator organism of fecal contamination. To achieve this, we first evaluated a number of current gene targets (24,25,26,27) and Q-PCR assays for E. coli. Furthermore, we expand their use by developing corresponding reverse transcription-quantitative PCR (RT-Q-PCR) assays to detect mRNA and explore their use as indicators of viable organisms.…”

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confidence: 99%

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

Abberton

Bereschenko

Wielen

et al. 2016

Appl Environ Microbiol

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Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T 90 (time taken for a reduction in cell number of 1 log 10 unit) values ranging from 17.4 ؎ 1.8 to 149 ؎ 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm ؊2 ; BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli. The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. IMPORTANCEThe provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely. S afe, clean drinking water is the foundation of society. However, even in developed countries, drinking water quality failures occur and have considerable public health impact. In particular, microbiological quality failures can be a significant threat to the supply of drinking water. The exact cause of these water quality malfunctions can be difficult to determine. In public water supplies, inefficient water treatment of the source could result in unwanted micr...

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“…Of 20 µL of each DNA solution, 5 µL was used for real-time quantitative PCR. We used the primers (PCR product size, 130 bp) designed for the single-copy gene uidA to detect chromosomal DNA (Chern et al, 2009). In addition, we designed primers (5′-CCACATCGAGGAAGAAAACG-3′ and 5′-CACGAT-AGAGCACCCGGTAT-3′; PCR product size, 91 bp) for repA and primers (5′-AATGCCATGTGGTCAAAGGT-3′ and 5′-CGATGCAGTCCTGTATGTGC-3′; PCR product size, 99 bp) for repC by using Primer3 (http://bioinfo.ut.ee/ primer3-0.4.0/) and used them to detect the plasmid pHRP311.…”

Section: Fig 2 Distribution Of Delta Cq Valuesmentioning

confidence: 99%

Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of <i>Escherichia coli</i>

Takahashi

Nishida

2015

J. Gen. Appl. Microbiol.

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None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.cillin (an inhibitor of peptidoglycan synthesis) was shown to generate giant protoplasts (Kuroda et al., 1998;Kusaka, 1967;Nakamura et al., 2011).Generally, DNA duplicates before cell division, and it is distributed to each cell during cell division. Most bacteria undergo symmetric binary fission. During the growth of spheroplasts, although cell division was not observed, DNA replication was, indeed, observed (Kuroda et al., 1998;Nakamura et al., 2011). However, it is uncertain whether plasmid DNA replicates during spheroplast growth. Several factors influence the synchronous regulation between chromosome and plasmid (Nordström and Dasgupta, 2006).In this study, we measured the chromosomal and plasmid DNA of E. coli spheroplasts by using real-time quantitative PCR, in order to elucidate the change in copy numbers of chromosomal and plasmid DNA during the growth of spheroplasts. Materials and MethodsPreparation of giant spheroplasts from E. coli. Giant spheroplasts were prepared using a modified version of the method previously described by Kusaka (1967) and the spheroplast incubation method reported by Kuroda et al. (1998). E. coli SCS1 (Stratagene) cells, which was transformed with the plasmid pHRP311 (14 kbp) with an RSF1010 replicon, were grown on marine broth agar (Difco) containing streptomycin under aerobic conditions. The harvested cells (0.003 g) were suspended in a buffer (1 mL) consisting of 0.1 M Tris-HCl (pH 7.6) and 0.3 M sucrose. Lysozyme (Wako) (200 µg/mL) was added to the cell suspension, and the suspension was incubated at 37°C for 15 min. After the suspension was divided into 2 aliquots (500 µL each), the cells were harvested (centrifugation Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli (Received August 8, 2015; Accepted September 9, 2015) Sawako Takahashi and Hiromi Nishida* Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Japan We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts. Biotechnology

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Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR

Cited by 56 publications

References 45 publications

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

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

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of <i>Escherichia coli</i>

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