Depuration processes affect the Vibrio community in the microbiota of the Manila clam, Ruditapes philippinarum

Abstract

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.

Combining Culture-Dependent and Culture-Independent Methods: New Methodology Insight on the Vibrio Community of Ruditapes philippinarum

“…Clams were processed as described in a previous study of Zampieri et al [19], with the addition of a microbiological analysis performed using a selective medium coupled with the temperature of incubation required for the detection of the main human pathogenic Vibrio species. Briefly, clams were scrubbed under running potable water and the shell cleaned with ethanol 100%.…”

“…For the microbial community of homogenates, DNA was extracted using a DNeasy ® PowerSoil ® Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. As described in a previous study [19], a mock community DNA sample was included into the sequencing libraries analysis as an internal control. Subsequently, each DNA sample was diluted for Illumina library preparation, performed on 16S rRNA, recA and pyrH genes.…”

“…For this purpose, in Vibrio research, next-generation sequencing (NGS) technology, a PCR-dependent method, has been applied to investigate Vibrio biodiversity in environmental and seafood samples. In particular, NGS analysis conducted on 16S rRNA combined with the sequencing of housekeeping genes such as heat shock protein 60 (hsp60) and recA-pyrH allowed for the identification of Vibrio species that naturally contaminate water, oyster, and Manila clam samples [17][18][19]. Moreover, this approach was used to successfully identify potential human pathogenic species such as V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus.…”

“…Moreover, amplicon-based methods present an intrinsic bias in the amplification step [22]. Designing inclusive universal primers located in regions more informative than 16S for species/strains' identification is challenging, and only parts of species/strains are successfully amplified [17][18][19]. The PCR bias can be overcome by using a shotgun metagenomics approach.…”

“…Following this idea, the objective of this study was to explore the Vibrio community composition of Ruditapes philippinarum microbiota by combining culturedependent and -independent methods. Specifically, DNA metabarcoding, developed in a previous study [19], was applied to evaluate and compare the accuracy of isolation and discrimination of Vibrio species identified on homogenate clam samples and on their culture-derived samples plated on marine agar (MA), thiosulfate-citrate-bile salts-sucrose agar (TCBS) and CHROMagar Vibrio (CV) media. On a subset of these culture-derived clam samples, shotgun metagenomics was applied in order to enhance the knowledge of the Vibrio spp.…”