Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Tissue preparation and nucleic acid purification influences the outcome of Pacific oyster (Crassostrea gigas) microbiome studies (#19)

Erandi Pathirana 1 , Richard Whittington 1 , Alison Tweedie 1 , Paul Hick 1
  1. Farm Animal Health, Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camden, NSW, Australia

Pacific oysters harbour a rich bacterial community of which a substantial proportion is non-cultivable. Bacterial DNA extraction in oyster microbiome studies faces many limitations. Different sampling techniques and nucleic acid extraction methods were tested to optimize the bacterial DNA yield and to accurately determine the microbiome associated with different oyster tissues.

Samples of haemolymph, swab samples and tissue samples of gills, gut and adductor-muscle were collected from Pacific oysters grown in the Georges River, NSW (n=10). Selected samples were enriched for bacterial DNA. Nucleic acids were extracted using three different commercial extraction kits. Bacterial DNA yields were assessed by a real-time PCR assay targeting the 16S rRNA gene. Bacterial community compositions were identified by high-throughput sequencing of the V1-V3 regions of the 16S rRNA gene. The relative abundance of bacterial phyla and diversity of OTUs were calculated for each sample. Beta diversity of bacterial communities obtained after different tissue preparation strategies were analysed.

Phylum Proteobacteria dominated in all tissue types except gut tissue where phylum Fusobacteria was abundant. Bacteroidetes, Spirochaetes were among the other abundant phyla. Beta diversity of bacterial community structure in different tissue types were significantly different. Higher bacterial diversity and bacterial DNA yield were noted in gill tissues compared to gut tissues (p<0.05). The highest bacterial DNA yield was obtained with the QIAamp® DNA microbiome kit extracts despite the lower bacterial diversity compared to EZNA Mollusc DNA kit extracts (p<0.05).

Apart from tissue specificity of the oyster microbiome, the tissue preparation strategy affects the quantity and diversity of bacteria identified in microbiome studies of the Pacific oyster. A clearly defined and fit-for-purpose sample preparation strategy is required to accurately identify the oyster microbiome.