Deciphering intra-species bacterial diversity of meat and seafood spoilage microbiota using gyrB amplicon sequencing: A comparative analysis with 16S rDNA V3-V4 amplicon sequencing

Abstract

Meat and seafood spoilage ecosystems harbor extensive bacterial genomic diversity that is mainly found within a small number of species but within a large number of strains with different spoilage metabolic potential. To decipher the intraspecies diversity of such microbiota, traditional metagenetic analysis using the 16S rRNA gene is inadequate. We therefore assessed the potential benefit of an alternative genetic marker, gyrB, which encodes the subunit B of DNA gyrase, a type II DNA topoisomerase. A comparison between 16S rDNA-based (V3-V4) amplicon sequencing and gyrB-based amplicon sequencing was carried out in five types of meat and seafood products, with five mock communities serving as quality controls. Our results revealed that bacterial richness in these mock communities and food samples was estimated with higher accuracy using gyrB than using16S rDNA. However, for Firmicutes species, 35% of putative gyrB reads were actually identified as sequences of a gyrB paralog, parE, which encodes subunit B of topoisomerase IV; we therefore constructed a reference database of published sequences of both gyrB and pare for use in all subsequent analyses. Despite this co-amplification, the deviation between relative sequencing quantification and absolute qPCR quantification was comparable to that observed for 16S rDNA for all the tested species. This confirms that gyrB can be used successfully alongside 16S rDNA to determine the species composition (richness and evenness) of food microbiota. The major benefit of gyrB sequencing is its potential for improving taxonomic assignment and for further investigating OTU richness at the subspecies level, thus allowing more accurate discrimination of samples. Indeed, 80% of the reads of the 16S rDNA dataset were represented by thirteen 16S rDNA-based OTUs that could not be assigned at the species-level. Instead, these same clades corresponded to 44 gyrB-based OTUs, which differentiated various lineages down to the subspecies level. The increased ability of gyrB-based analyses to track and trace phylogenetically different groups of strains will generate improved resolution and more reliable results for studies of the strains implicated in food processes.

Combination of high pressure treatment at 500 MPa and biopreservation with aLactococcus lactisstrain for lowering the bacterial growth during storage of diced cooked ham with reduced nitrite salt

“…Total genomic DNA was extracted from the bacterial pellet as described previously (Poirier et al, 2018) using the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc., Carlsbad, USA) and the High Pure PCR Template Preparation kit (Roche Diagnostics Ltd, Burgess Hill, West Sussex, UK). For each sample, both DNA extracts were pooled.…”

“…For each sample, both DNA extracts were pooled. Then, this DNA sample was used as template for three independent amplifications using either the 16S V3-V4 region of the rRNA encoding gene or an internal 280 bp fragment of the Gyrase B subunit encoding gene gyrB, as described previously (Poirier et al, 2018). All PCRs were performed in triplicate.…”

MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction

“…While shotgun sequencing of the whole community (metagenomics) can provide a richer description of the functions in a community, HTAS remains a more efficient tool for comparing the species diversity of a large number of community samples. Despite the extensive use of HTAS for interspecies ecological diversity studies, few investigations have utilised HTAS for intraspecies analysis [9,10]. As 16S rRNA amplicons are too highly conserved to estimate microbial within-species diversity, other target gene candidates need to be considered in order to sufficiently discern intraspecies sequence variation.…”

MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction

“…HTAS allows for more in-depth, higher resolution sequencing of a specific part of the genome for diversity estimates compared to shotgun sequencing, but at the cost of potential sequence errors (Gohl et al, 2016). Despite the extensive use of HTAS for interspecies ecological diversity studies, to our knowledge few investigations have utilised HTAS for intraspecies analysis (Poirier et al, 2018;Kinoti et al, 2017). As 16S rRNA amplicons are too highly conserved to estimate microbial within-species diversity, other target gene candidates would need to be considered in order to sufficiently discern intraspecies sequence variation.…”