Development of a mreB-targeted real-time PCR method for the quantitative detection of Vibrio harveyi in seawater and biofilm from aquaculture systems

Abstract

Vibrio harveyi is a particularly problematic Gram-negative bacterium because it can form biofilms on aquaculture facility surfaces, leading to resistance of bacteria against antibiotics and water sanitizers. A SYBR Green I quantitative real-time PCR method was developed to detect V. harveyi directly from environmental samples, including seawater and biofilm. Specific primers targeting the mreB gene were designed. The exclusivity and inclusivity of the newly designed primers were evaluated using a panel of 85 bacteria: 58 V. harveyi from multiples origins and 27 non-V. harveyi isolates, and compared with two pairs of primers targeting the topA and toxR genes that were designed previously. All sets of primers were able to distinguish V. harveyi from closely related species belonging to the Harveyi clade. However, the mreB primers showed better inclusivity and were thus used to develop the real-time PCR assay. A quantification curve was obtained from pure culture of V. harveyi and exhibited excellent efficacy with detection levels as low as 5 genome copies in the PCR reaction. After selection of the extraction kit allowing the best DNA quantity and purity, validation was performed on both seawater and biofilm samples collected from a fish farm. The presence of inhibitors in the DNA templates was evaluated and a 10-fold dilution of template DNA was recommended in order to avoid their effects. The assay was able to detect V. harveyi from environmental samples, confirming the validity of the method. This real-time PCR method will help to evaluate the dynamics of V. harveyi in aquaculture facilities. Suitable prophylactic control measures could be designed using this method, instead of the use of curative methods such as antibiotics

Rapid Identification of Vibrio Species of the Harveyi Clade Using MALDI-TOF MS Profiling With Main Spectral Profile Database Implemented With an In-House Database: Luvibase

“…These spectra were first compared to the MSPs included in the Bruker ver.9.0.0.0 database. The bacterial identifications previously obtained in the study of Mougin et al (2020) were then compared with the MALDI-TOF MS profile-based bacterial identifications. The matching rates were 100% at the genus level and only 75.3% at the species level, assuming a log-score of at least 2.0 required for probable species identification ( Table 1 ).…”

“…A total of 85 strains were used in this study ( Table 1 ). These strains were described and identified by PCR, sequencing of house-keeping genes or whole genome sequencing in a previous study ( Mougin et al, 2020 ). Briefly, (i) 38 collection strains were obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG), the German Collection of Microorganisms and Cell Cultures (DSMZ), the Spanish Type Culture Collection (CECT), and the Institut Pasteur Collection (CIP), (ii) 35 isolates were obtained from a fish farm (Aquanord-Ichtus, Gravelines, France) raising seabass Dicentrarchus labrax and (iii) 12 isolates were isolated by the French Research Institute for the Exploitation of the Sea (IFREMER) from oysters ( Crassostrea gigas ) and abalone ( Haliotis tuberculata ).…”

“…More specifically, our objective was to develop a method for the accurate identification of pathogens obtained from moribund abalone ( Haliotis tuberculata ), seabass ( Dicentrarchus labrax ), and oyster ( Crassostrea gigas ) from aquaculture farms. These strains have been previously identified using PCR and sequencing by Mougin et al (2020) . Given that the MALDI BioTyper was not adequate for species identification, we constructed an in-house database, named Luvibase.…”

Development of real‐time fluorescence saltatory rolling circle amplification for rapid detection of Vibrio parahaemolyticus in seafood

“…parahaemolyticus and is described in many other Vibrio spp. (Chen & Ge, 2010; Letchumanan et al ., 2014; Mougin et al ., 2020). The coding sequence of toxR related to the membrane ‘tether’ region possesses significant heterogeneity (Chen & Ge, 2010); thus, V .…”

“…As an essential role in modulating bacterial pathogenesis and persistence (Chen et al, 2018;Zhang et al, 2018), the toxR gene exists in all V. parahaemolyticus and is described in many other Vibrio spp. (Chen & Ge, 2010;Letchumanan et al, 2014;Mougin et al, 2020). The coding sequence of toxR related to the membrane 'tether' region possesses significant heterogeneity (Chen & Ge, 2010); thus, V. parahaemolyticus-specific assays targeting toxR gene have been successfully implemented in many studies, such as toxR-based PCR assays (Taiwo et al, 2017;Ling et al, 2020) and LAMP methods (Bonnin-Jusserand et al, 2019;Kampeera et al, 2019), confirming the feasibility of toxR as a reliable species-specific target gene.…”