Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water

Wilhartitz, Inés C.; Mach, Robert L.; Teira, Eva; Reinthaler, Thomas; Herndl, Gerhard J.; Farnleitner, Andreas H.

doi:10.1111/j.1365-2672.2007.03319.x

Cited by 37 publications

(30 citation statements)

References 53 publications

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“…The newly developed protocol is based on the CARD-FISH protocol described by Wilhartitz et al (37), with modifications. Samples were fixed with 4% PFA and filtered on CB04 filters (black; Ø, 25 mm; pore size, 0.4 m; AES Chemunex).…”

Section: Methodsmentioning

confidence: 99%

“…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). The rRNAtargeted oligonucleotide probe is labeled with horseradish peroxidase (HRP).…”

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confidence: 99%

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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: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…The non-EUB-338 probe (4) was also included as an additional control to monitor nonspecific probe binding. For V. souniana, cell wall permeabilization was required (by incubation with proteinase K 4U/ml at 37°C for 1 h; Sigma, St. Louis, MO) due to its protein-rich cell wall (70,74).…”

Section: Methodsmentioning

confidence: 99%

Assessment of the Diversity, Abundance, and Ecological Distribution of Members of Candidate Division SR1 Reveals a High Level of Phylogenetic Diversity but Limited Morphotypic Diversity

Davis

Youssef

2009

Appl Environ Microbiol

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We used a combination of 16S rRNA gene clone library surveys, quantitative PCR (qPCR) analysis, and fluorescent in situ hybridization to investigate the diversity, abundance, and distribution of members of candidate division SR1 in multiple habitats. Using SR1-specific 16S rRNA gene primers, we identified multiple novel SR1 lineages in four different anaerobic environments: sediments from Zodletone Spring, a sulfide-and sulfur-rich spring in southwestern Oklahoma; inner layers of microbial mats obtained from Sperm Pool, a high-temperature, low-pH pool (55°C, pH 2.5) in Yellowstone National Park; fresh bovine ruminal contents; and anaerobic freshwater pond sediments (Duck Pond) in Norman, Oklahoma. qPCR analysis indicated that SR1 members constitute a small fraction (<0.01%) of the microbial communities in Duck Pond and ruminal samples but constitute a significant fraction (11.6 and 48.7%) of the total number of bacterial 16S rRNA genes in Zodletone Spring and the inner layers of Sperm Pool microbial mat samples, respectively. By using SR1-specific fluorescent probes, filamentous cells were identified as the sole SR1 morphotype in all environments examined, with the exception of Sperm Pool, where a second bacillus morphotype was also identified. Using a full-cycle 16S rRNA approach, we show that each of these two morphotypes corresponds to a specific phylogenetic lineage identified in the Sperm Pool clone library. This work greatly expands the intralineage phylogenetic diversity within candidate division SR1 and provides valuable quantification and visualization tools that could be used for investigating the ecological roles, dynamics, and genomics of this as-yet-uncultured bacterial phylum.

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“…1995 ;Pernthaler et al . 2002 ;Wilhartitz et al ., 2007 ;Eickhorst and Tippkötter, 2008 ;Shiraishi et al ., 2008 ;Schramm and Amann 2008 RISA/ribosomal intergenic spacer Fast and large numbers of samples possible. Automatic approach possible.…”

Section: Referencementioning

confidence: 95%

Molecular methods for studying biocorrosion

Graeber

Boehm

Kuever

2014

Understanding Biocorrosion

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Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water

Cited by 37 publications

References 53 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

Assessment of the Diversity, Abundance, and Ecological Distribution of Members of Candidate Division SR1 Reveals a High Level of Phylogenetic Diversity but Limited Morphotypic Diversity

Molecular methods for studying biocorrosion

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