Laser scanning detection of FISH-labelled Escherichia coli from water samples

Lepeuple, A; Delabre, Karine; Gilouppe, S.; Intertaglia, Laurent; Roubín, Mirian Raposeiras

doi:10.2166/wst.2003.0179

Cited by 13 publications

(7 citation statements)

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“…Our results are consistent with previous studies which demonstrated that FISH gives a too weak signal for SPC (24,34) and that CARD-FISH is a methodical improvement to increase the fluorescence signal (30). The choice of an appropriate amplification time during the CARD-FISH protocol is especially important for increasing the signal.…”

Section: Discussionsupporting

confidence: 92%

“…This fact is an important advantage over flow cytometry and fluorescence microscopy, for which, in contrast to solid-phase cytometry, large volumes need to be concentrated for detection of rare events. Unfortunately, previous studies (24,34) have shown that the fluorescence signals of standard FISH-labeled microorganisms were too weak to be detected with solid-phase cytometry. Catalyzed reporter deposition-FISH (CARD-FISH) is one methodical improvement to increase the signal intensity of microorganisms (30,33,37).…”

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confidence: 98%

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Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

Schauer¹,

Sommer

Farnleitner

et al. 2012

Appl Environ Microbiol

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A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 ؋ 10 1 cells ml ؊1 in the large saline lake Neusiedler See to 5.6 ؋ 10 4 cells ml ؊1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.V ibrio cholerae and Vibrio mimicus are important human pathogens and two very closely related species. Only recently, their classification as two different species has been confirmed (35). V. cholerae comprises more than 200 serotypes, but only serotypes O1 and O139 cause the severe diarrheal disease cholera (9, 32). Interestingly, several V. mimicus isolates carrying cholera toxin genes (ctxAB) have also been identified. It was suggested that horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae have generated pathogenic V. mimicus strains carrying cholera toxin genes (35). All other V. cholerae serotypes, summarized as non-O1/non-O139, and V. mimicus strains are usually associated with less-severe gastrointestinal infections as well as blood, wound, and ear infections (7,16,28,35). Besides their role as important human pathogens, both organisms are found worldwide in brackish water, coastal areas, and estuarine environments (8,35) and are therefore considered to be natural components of the bacterial community in aquatic ecosystems.Rapid and precise methods for detection and quantification of pathogenic bacteria are fundamental for monitoring and risk assessment in regard to the use of water as well as for studying their ecology in the environment. For quantification of the V. cholerae/V. mimicus clade, various methods have been developed during the last decades. Culture-based techniques for detecting these pathogens, followed by biochemical confirmation via API 20E or PCR, are still regularly used (5, 8). These techniques often include an enrichment step with alkaline peptone water and can then be used quantitatively only as laborious most-probable-number (MPN) techniques. In addition, they cannot be applied when Vibrio cell...

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 98%

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

Schauer¹,

Sommer

Farnleitner

et al. 2012

Appl Environ Microbiol

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“…Although the PCR method is considered to be more sensitive than either FISH or the culture methods, more H. pylori ‐positive samples were obtained by the FISH technique than by culture or PCR analysis, confirming previous reports . PCR analysis allowed for the direct detection of H. pylori DNA in only three raw samples.…”

Section: Resultssupporting

confidence: 83%

Specific Detection of Cultivable Helicobacter pylori Cells from Wastewater Treatment Plants

Moreno

Ferrús

2012

Helicobacter

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Background Helicobacter pylori is present in surface water and wastewater, and biofilms in drinking water systems have been reported as possible reservoirs of H. pylori. However, its ability to survive in an infectious state in the environment is hindered because it rapidly loses its cultivability. The aim of this study was to determine the presence of cultivable and therefore viable H. pylori in wastewater treatment plants to understand the role of wastewater in the pathogen's transmission. Materials and Methods A modified filter technique was used to obtain a positive H. pylori culture, and specific detection of this pathogen was achieved with FISH and PCR techniques. Results A total of six positive H. pylori cultures were obtained from the water samples, and molecular techniques positively identified H. pylori in 21 culture‐negative samples. Conclusions The combination of a culturing procedure after sample filtration followed by the application of a molecular method, such as PCR or FISH, provides a specific tool for the detection, identification, and direct visualization of cultivable and therefore viable H. pylori cells from complex mixed communities such as water samples.

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“…12 The application of flow cytometry (FCM) provides a more rapid and reliable quantification than epifluorescence microscopy, ensuring accuracy and precision in enumeration. 13−15 However, previous studies have demonstrated that FISH gives a signal that is too weak for flow-cytometric measurements, 16,17 which greatly limits the utilization of FISH for identification of microbes with slow growth rates (e.g., anammox bacteria) or low ribosome contents. 18 Catalyzed reporter deposition (CARD)-FISH is a methodical improvement in fluorescence signal enhancement 19 that has been applied to the detection and quantification of microorganisms characterized by low ribosome contents or rather low abundance, such as marine microorganisms, biofilms, and benthic detritus.…”

Section: ■ Introductionmentioning

confidence: 99%

Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization

Zhu

Wang

Yuan

et al. 2019

Environ. Sci. Technol.

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The quantification of anammox bacteria is crucial to manipulation and management of anammox biosystems. In this study, we proposed a protocol specifically optimized for quantification of anammox bacteria abundance in anammox sludge samples using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and flow cytometry (FCM) in combination (Flow-CARD-FISH). We optimized the pretreatment procedures for FCM-compatibility, as well as the permeabilization, hybridization and staining protocols of the CARD-FISH. The developed method was compared with other methods for specific bacteria quantification (standard FISH, 16S rRNA sequencing and quantitative polymerase chain reaction). Anammox sludge samples could be disaggregated effectively by sonication (specific energy of 90 kJ·L–1 with MLVSS of 3–5 g·L–1) with the mixed ionic and nonionic dispersants Triton X-100 (5%) and sodium pyrophosphate (10 mM). Lysozyme treatment for permeabilizing bacterial cell walls and H2O2 incubation for completely quenching endogenous peroxidase of anammox sludges were essential to fluorescence enhancement and false positive signals control, respectively. Horseradish peroxidase molecules labeling at 20 °C for 12 h and the fluorescent tyramide labeling at 25 °C for 30 min with a fluorescent substrate concentration of 1:50 maintained the balance between increasing the signal and preventing nonspecific binding. Flow-CARD-FISH results showed that anammox bacteria absolute abundance in two different sludge samples were (2.31 ± 0.01) × 107 and (1.20 ± 0.06) × 107 cells·mL–1, respectively, with the relative abundances of 36.7 ± 4.1% and 26.5 ± 3.7%, respectively, comparable with those of qPCR and 16S rRNA sequencing analysis. The enhanced fluorescence signals induced by CARD-FISH combined with the high quantitative fluorescence sensitivity of FCM provide a rapid and sensitive method that yields accurate quantification results that will be valuable in future studies of microbial community determination.

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Laser scanning detection of FISH-labelled Escherichia coli from water samples

Cited by 13 publications

References 0 publications

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

Specific Detection of Cultivable Helicobacter pylori Cells from Wastewater Treatment Plants

Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization

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