An electrochemical RNA hybridization assay for detection of the fecal indicator bacterium Escherichia coli

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

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Escherichia coli is not a suitable fecal indicator to assess water fecal contamination by otters

Brazilian Journal of Biology ◽

10.1590/1519-6984.167279 ◽

2017 ◽

Vol 78 (1) ◽

pp. 155-159 ◽

Cited By ~ 2

Author(s):

M. Oliveira ◽

D. Freire ◽

N. M. Pedroso

Keyword(s):

Escherichia Coli ◽

Fecal Contamination ◽

Microbiological Quality ◽

Aquatic Environments ◽

Pathogenic Microorganisms ◽

Fecal Indicator ◽

Fecal Samples ◽

E Coli ◽

Paulo State

Abstract The detection of pathogenic microorganisms in aquatic environments is extremely relevant in terms of public health. As these laboratorial methodologies are usually difficult, expensive and time-consuming, they are frequently replaced by the assessment of fecal indicator bacteria, such as Escherichia coli. This study aimed to assess the presence of E. coli in fecal samples from Neotropical otters, to evaluate its potential as fecal indicator to be applied to the determination of water microbiological quality in areas where otters’ populations are high. Twenty-six otter fecal samples, collected in Alto Paranapanema river basin, São Paulo State, Brazil, were analyzed for the presence of E. coli, using conventional bacteriological methods. Only 8 scat samples (30%) were E. coli positive, indicating that this microorganism is not a suitable fecal indicator to assess water fecal contamination by Neotropical otters, and should not be used to infer the presence of otter related pathogens in waters.

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Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

Applied and Environmental Microbiology ◽

10.1128/aem.01370-08 ◽

2008 ◽

Vol 74 (24) ◽

pp. 7463-7470 ◽

Cited By ~ 19

Author(s):

Daniel Thieme ◽

Peter Neubauer ◽

Dietrich H. Nies ◽

Gregor Grass

Keyword(s):

Escherichia Coli ◽

Alkaline Phosphatase ◽

Time Course ◽

Copper Ions ◽

Copper Resistance ◽

Hybridization Assay ◽

Target Mrna ◽

Cell Extracts ◽

Sandwich Hybridization ◽

Sandwich Hybridization Assay

ABSTRACT Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.

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Poikilothermic Animals as a Previously Unrecognized Source of Fecal Indicator Bacteria in a Backwater Ecosystem of a Large River

Applied and Environmental Microbiology ◽

10.1128/aem.00715-18 ◽

2018 ◽

Vol 84 (16) ◽

Cited By ~ 6

Author(s):

Christina Frick ◽

Julia Vierheilig ◽

Rita Linke ◽

Domenico Savio ◽

Horst Zornig ◽

...

Keyword(s):

Escherichia Coli ◽

Clostridium Perfringens ◽

Fecal Indicator Bacteria ◽

Indicator Bacteria ◽

Primary Sources ◽

Fecal Indicator ◽

Content Type ◽

E Coli ◽

High Concentrations ◽

Intestinal Enterococci

ABSTRACTQuantitative information regarding the presence ofEscherichia coli, intestinal enterococci, andClostridium perfringensin poikilotherms is notably scarce. Therefore, this study was designed to allow a systematic comparison of the occurrence of these standard fecal indicator bacteria (SFIB) in the excreta of wild homeothermic (ruminants, boars, carnivores, and birds) and poikilothermic (earthworms, gastropods, frogs, and fish) animals inhabiting an alluvial backwater area in eastern Austria. With the exception of earthworms, the average concentrations ofE. coliand enterococci in the excreta of poikilotherms were equal to or only slightly lower than those observed in homeothermic excreta and were 1 to 4 orders of magnitude higher than the levels observed in the ambient soils and sediments. Enterococci reached extraordinarily high concentrations in gastropods. Additional estimates of the daily excreted SFIB (E. coliand enterococcus) loads (DESL) further supported the importance of poikilotherms as potential pollution sources. The newly established DESL metric also allowed comparison to the standing stock of SFIB in the sediment and soil of the investigated area. In agreement with its biological characteristics, the highest concentrations ofC. perfringenswere observed in carnivores. In conclusion, the long-standing hypothesis that only humans and homeothermic animals are primary sources of SFIB is challenged by the results of this study. It may be necessary to extend the fecal indicator concept by additionally considering poikilotherms as potential important primary habitats of SFIB. Further studies in other geographical areas are needed to evaluate the general significance of our results. We hypothesize that the importance of poikilotherms as sources of SFIB is strongly correlated with the ambient temperature and would therefore be of increased significance in subtropical and tropical habitats and water resources.IMPORTANCEThe current fecal indicator concept is based on the assumption that the standard fecal indicator bacteria (SFIB)Escherichia coli, intestinal enterococci, andClostridium perfringensmultiply significantly only in the guts of humans and other homeothermic animals and can therefore indicate fecal pollution and the potential presence of pathogens from those groups. The findings of the present study showed that SFIB can also occur in high concentrations in poikilothermic animals (i.e., animals with body temperatures that vary with the ambient environmental temperature, such as fish, frogs, and snails) in an alluvial backwater area in a temperate region, indicating that a reconsideration of this long-standing indicator paradigm is needed. This study suggests that poikilotherms must be considered to be potential primary sources of SFIB in future studies.

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Salmonella enterica Serovar Typhimurium and Escherichia coli Survival in Estuarine Bank Sediments

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph15112597 ◽

2018 ◽

Vol 15 (11) ◽

pp. 2597 ◽

Cited By ~ 2

Author(s):

Mahbubul Siddiqee ◽

Rebekah Henry ◽

Rebecca Coulthard ◽

Christelle Schang ◽

Richard Williamson ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Growth Patterns ◽

Estuarine Sediments ◽

Human Pathogens ◽

Indicator Organisms ◽

Fecal Indicator ◽

E Coli ◽

Serovar Typhimurium

Estuarine bank sediments have the potential to support the survival and growth of fecal indicator organisms, including Escherichia coli. However, survival of fecal pathogens in estuarine sediments is not well researched and therefore remains a significant knowledge gap regarding public health risks in estuaries. In this study, simultaneous survival of Escherichia coli and a fecal pathogen, Salmonella enterica serovar Typhimurium, was studied for 21 days in estuarine bank sediment microcosms. Observed growth patterns for both organisms were comparable under four simulated scenarios; for continuous-desiccation, extended-desiccation, periodic-inundation, and continuous-inundation systems, logarithmic decay coefficients were 1.54/day, 1.51/day, 0.14/day, and 0.20/day, respectively, for E. coli, and 1.72/day, 1.64/day, 0.21/day, and 0.24/day for S. Typhimurium. Re-wetting of continuous-desiccated systems resulted in potential re-growth, suggesting survival under moisture-limited conditions. Key findings from this study include: (i) Bank sediments can potentially support human pathogens (S. Typhimurium), (ii) inundation levels influence the survival of fecal bacteria in estuarine bank sediments, and (iii) comparable survival rates of S. Typhimurium and E. coli implies the latter could be a reliable fecal indicator in urban estuaries. The results from this study will help select suitable monitoring and management strategies for safer recreational activities in urban estuaries.

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The effect of pore size in an ultrasensitive DNA sandwich-hybridization assay for the Escherichia coli O157:H7 gene based on the use of a nanoporous alumina membrane

Microchimica Acta ◽

10.1007/s00604-017-2530-7 ◽

2017 ◽

Vol 184 (12) ◽

pp. 4835-4844 ◽

Cited By ~ 9

Author(s):

Weiwei Ye ◽

Tian Chen ◽

Yijie Mao ◽

Feng Tian ◽

Peilong Sun ◽

...

Keyword(s):

Escherichia Coli ◽

Pore Size ◽

Hybridization Assay ◽

Escherichia Coli O157 ◽

Alumina Membrane ◽

Nanoporous Alumina ◽

Sandwich Hybridization ◽

Sandwich Hybridization Assay

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Occurrence of Escherichia coli and Enterococci in Cladophora (Chlorophyta) in Nearshore Water and Beach Sand of Lake Michigan

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4714-4719.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4714-4719 ◽

Cited By ~ 194

Author(s):

Richard L. Whitman ◽

Dawn A. Shively ◽

Heather Pawlik ◽

Meredith B. Nevers ◽

Muruleedhara N. Byappanahalli

Keyword(s):

Escherichia Coli ◽

Lake Michigan ◽

Dry Weight ◽

Point Sources ◽

Indicator Bacteria ◽

Beach Sand ◽

Cladophora Glomerata ◽

Strongly Correlated ◽

Fecal Indicator ◽

E Coli

ABSTRACT Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (± standard errors) of 5.3 (± 4.8) and 4.8 (± 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R 2 = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4°C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.

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