Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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Molecular detection of Helicobacter pylori in a large Mediterranean river, by direct viable count fluorescent in situ hybridization (DVC-FISH)

Journal of Water and Health ◽

10.2166/wh.2014.171 ◽

2014 ◽

Vol 12 (4) ◽

pp. 868-873 ◽

Cited By ~ 9

Author(s):

Ilias Tirodimos ◽

Mattheos Bobos ◽

Evangelos Kazakos ◽

Anna-Bettina Haidich ◽

Theodore Dardavessis ◽

...

Keyword(s):

Helicobacter Pylori ◽

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Potable Water ◽

Molecular Detection ◽

Viable Count ◽

Potential Health ◽

Viable Cells ◽

H Pylori

Although the precise route and mode of transmission of Helicobacter pylori are still unclear, molecular methods have been applied for the detection of H. pylori in environmental samples. In this study, we used the direct viable count fluorescent in situ hybridization (DVC-FISH) method to detect viable cells of H. pylori in the River Aliakmon, Greece. This is the longest river in Greece, and provides potable water in metropolitan areas. H. pylori showed positive detection for 23 out of 48 water samples (47.9%), while no seasonal variation was found and no correlation was observed between the presence of H. pylori and indicators of fecal contamination. Our findings strengthen the evidence that H. pylori is waterborne while its presence adds to the potential health hazards of the River Aliakmon.

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Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

Biogeosciences ◽

10.5194/bg-8-2075-2011 ◽

2011 ◽

Vol 8 (8) ◽

pp. 2075-2088 ◽

Cited By ~ 8

Author(s):

C. Borrelli ◽

A. Sabbatini ◽

G. M. Luna ◽

M. P. Nardelli ◽

T. Sbaffi ◽

...

Keyword(s):

In Situ Hybridization ◽

Benthic Foraminifera ◽

Fluorescent In Situ Hybridization ◽

Microscopic Analysis ◽

Technical Note ◽

Physiological Status ◽

Hybridization Technique ◽

Marine Biota ◽

Classical Studies

Abstract. Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

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