Dataset Information

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.

SUBMITTER: Poirier S

PROVIDER: S-EPMC6155546 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

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.

Poirier Simon S Rué Olivier O Peguilhan Raphaëlle R Coeuret Gwendoline G Zagorec Monique M Champomier-Vergès Marie-Christine MC Loux Valentin V Chaillou Stéphane S

PloS one 20180925 9

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 topoisomer ...[more]

PMID: 30252901

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Project description:BACKGROUND: Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. RESULTS: The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2?×?10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. CONCLUSION: This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies.

Project description:Microbial communities of the Arctic Ocean are poorly characterized in comparison to other aquatic environments as to their horizontal, vertical, and temporal turnover. Yet, recent studies showed that the Arctic marine ecosystem harbors unique microbial community members that are adapted to harsh environmental conditions, such as near-freezing temperatures and extreme seasonality. The gene for the small ribosomal subunit (16S rRNA) is commonly used to study the taxonomic composition of microbial communities in their natural environment. Several primer sets for this marker gene have been extensively tested across various sample sets, but these typically originated from low-latitude environments. An explicit evaluation of primer-set performances in representing the microbial communities of the Arctic Ocean is currently lacking. To select a suitable primer set for studying microbiomes of various Arctic marine habitats (sea ice, surface water, marine snow, deep ocean basin, and deep-sea sediment), we have conducted a performance comparison between two widely used primer sets, targeting different hypervariable regions of the 16S rRNA gene (V3-V4 and V4-V5). We observed that both primer sets were highly similar in representing the total microbial community composition down to genus rank, which was also confirmed independently by subgroup-specific catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) counts. Each primer set revealed higher internal diversity within certain bacterial taxonomic groups (e.g., the class Bacteroidia by V3-V4, and the phylum Planctomycetes by V4-V5). However, the V4-V5 primer set provides concurrent coverage of the archaeal domain, a relevant component comprising 10-20% of the community in Arctic deep waters and the sediment. Although both primer sets perform similarly, we suggest the use of the V4-V5 primer set for the integration of both bacterial and archaeal community dynamics in the Arctic marine environment.

Project description:Although several studies have focused on the dynamics of bacterial food community, little is known about the variability of batch production and microbial changes that occur during storage. The aim of the study was to characterize the microbial spoilage community of minced pork meat samples, among different food production and storage, using both 16S rRNA gene sequencing and classical microbiology. Three batches of samples were obtained from four local Belgian facilities (A-D) and stored until shelf life under food wrap (FW) and modified atmosphere packaging (MAP, CO2 30%/O2 70%), at constant and dynamic temperature. Analysis of 288 samples were performed by 16S rRNA gene sequencing in combination with counts of psychrotrophic and lactic acid bacteria at 22°C. At the first day of storage, different psychrotrophic counts were observed between the four food companies (Kruskal-Wallist test, p-value < 0.05). Results shown that lowest microbial counts were observed at the first day for industries D and A (4.2 ± 0.4 and 5.6 ± 0.1 log CFU/g, respectively), whereas industries B and C showed the highest results (7.5 ± 0.4 and 7.2 ± 0.4 log CFU/g). At the end of the shelf life, psychrotrophic counts for all food companies was over 7.0 log CFU/g. With metagenetics, 48 OTUs were assigned. At the first day, the genus Photobacterium (86.7 and 19.9% for food industries A and C, respectively) and Pseudomonas (38.7 and 25.7% for food companies B and D, respectively) were dominant. During the storage, a total of 12 dominant genera (>5% in relative abundance) were identified in MAP and 7 in FW. Pseudomonas was more present in FW and this genus was potentially replaced by Brochothrix in MAP (two-sided Welch's t-test, p-value < 0.05). Also, a high Bray-Curtis dissimilarity in genus relative abundance was observed between food companies and batches. Although the bacteria consistently dominated the microbiota in our samples are known, results indicated that bacterial diversity needs to be addressed on the level of food companies, batches variation and food storage conditions. Present data illustrate that the combined approach provides complementary results on microbial dynamics in minced pork meat samples, considering batches and packaging variations.

Dataset Information

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.

Publications

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.

Similar Datasets

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