Efficiency and specificity of CARD‐FISH probes in detection of marine vibrios

Abstract

Summary Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio‐associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture‐based methods (e.g. selection on TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially in microbial populations containing a fraction of ‘dormant’ cells (e.g. cells in the Viable but Non Culturable [VBNC] state). This problem can be overcome by using alternative culture‐free approaches such as Catalysed Reporter Deposition‐Fluorescence In situ Hybridization (CARD‐FISH). To select an efficient CARD‐FISH probe for detection of Vibrio spp. in environmental samples, we have assessed the most promising probes described in the literature by using both computer‐assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD‐FISH detection of marine vibrios in natural seawater.

Quantitative Detection of VBNC State Pseudomonas aeruginosa Contributing to Accurate Assessment of Microbial Inactivation in Drinking Water Disinfection

“…Various activity-based techniques have been developed to detect bacteria in the VBNC state, including measurements of respiratory and metabolic activity, fluorescent-based hybridization, and staining techniques based on cell membrane integrity [8][9][10]. However, these methods face limitations in their broad applicability.…”

“…While HPC methodologies have achieved a state of maturity, the application of qPCR in drinking water disinfection remains an evolving area. In the majority of studies, the pretreatment of bacteria with the dye propidium monoazide (PMA), wherein PMA can penetrate damaged cell membranes that hinder DNA amplification [10], was used for the detection of viable cells using qPCR. However, it is imperative to acknowledge that not all dead cells necessarily experience membrane damage; some may die due to internal DNA damage [12][13][14].…”

Harnessing the intragenomic variability of rRNA operons to improve differentiation of Vibrio species

“…As many marine and aquatic vibrios can be cultured in vitro, and a rough assessment of Vibrio community composition and dynamics can be carried out by using culture-based techniques (e.g., incubation on selective media such as TCBS agar or CHROMagar Vibrio). However, more complete information about community structure that includes both culturable and non-culturable (dormant) vibrios is obtainable with molecular tools such as CARD-FISH 19 and analysis of metagenomic DNA extracted from environmental samples. Moreover, the culturable vibrios can subsequently be purified and genotyped to reveal species identities.…”