Application of molecular tools to elucidate the microbiota of seafood

Abstract

The aim of this review is to present the methodologies currently applied to identify microbiota and pathogens transmitted to humans through seafood consumption, focusing on molecular techniques and pointing out their importance, advantages, disadvantages and applicability. Knowledge of available techniques allows researchers to identify which technique best fits their expectations. With such discernment, it will be possible to infer which disadvantages will be present and, therefore, not interfering with the final result. Two methodologies can be employed for this purpose, dependent and independent cultures. However, the dependent culture has certain limitations that can be solved through the independent cultivation techniques, such as PCR, PFGE and NGS, especially through the sequencing of the 16S rRNA region, providing a complete view of microbial diversity. These have revolutionized microbiological knowledge, mainly because they allow for the identification of uncultivable micro-organisms, which represent a substantial portion of total micro-organisms, making it possible to elucidate not yet described taxa which may display pathogenic potential, besides quantifying microbial communities, microbiota genetics, translated proteins and produced metabolites. In addition, transcriptomic and metabolomic techniques also allow for the evaluation of possible impacts that microbial communities may create in their environment, as well as the determination of potential pathogenicity to humans.

Characterization of the Spoilage Microbiota of Hake Fillets Packaged Under a Modified Atmosphere (MAP) Rich in CO2 (50% CO2/50% N2) and Stored at Different Temperatures

“…PCR assays (as the one used here for quantifying Photobacterium ) have many advantages as described elsewhere [34] but, as for culture methods, one of the weaknesses of PCR is that target sequences for primer design must be chosen before testing begins. By contrast, HTS is the most in-depth and unbiased method of obtaining genomic or metagenomic information [17,35]. Unlike PCR or microarrays, it does not require primer or probe design, it can be easily multiplexed, and the specificity and selectivity of the sequencing can be adjusted computationally after acquiring the data [36,37].…”

“…Unlike PCR or microarrays, it does not require primer or probe design, it can be easily multiplexed, and the specificity and selectivity of the sequencing can be adjusted computationally after acquiring the data [36,37]. High-throughput 16S rRNA gene sequencing is the most widely used technique for microbial diversity analysis and has been applied to various environments [31], including seafood [17]. It nevertheless has some pitfalls, mainly its limitation to differentiate at the species level as well as between live and dead microorganisms; furthermore, it cannot be used to determine the absolute number of microorganisms in a sample [15,16,17].…”

“…High-throughput 16S rRNA gene sequencing is the most widely used technique for microbial diversity analysis and has been applied to various environments [31], including seafood [17]. It nevertheless has some pitfalls, mainly its limitation to differentiate at the species level as well as between live and dead microorganisms; furthermore, it cannot be used to determine the absolute number of microorganisms in a sample [15,16,17].…”

“…High-throughput sequencing technology has become a widely used tool for the study of whole communities of prokaryotes in many niches [8], including fish stored under different conditions [9,10,11,12,13,14]. Nevertheless, this technique also has its own perils and pitfalls, most of which have already been summarized by Janda and Abbott [15], Ercolini [16] and, more recently, by Rodrigues et al [17].…”

Phylogenomics of expanding uncultured environmental Tenericutes provides insights into their pathogenicity and evolutionary relationship with Bacilli

“…The paucity of marine isolates currently available has limited further mechanistic insights. Using culture-independent highthroughput sequencing techniques, Tenericutes have been detected in the gut and gonad microbiomes of fish, sea star, oysters and mussel [5][6][7]. As seafood consumption rises [8], there are greater concerns about food safety and control.…”