Insights into Aquatic Biofilm Dynamics by High-Throughput Identification of Bacterial Isolates and Full-Length 16S rRNA Gene Surveys

Microbial biofilms form on all aquatic surfaces and can harbor pathogenic bacteria. In the aquaculture industry, Flavobacteria species can cause serious diseases and lead to high mortality. These disease outbreaks can reoccur on potential reservoir of these pathogens are biofilms. We characterized the biofilms in a commercial trout farm by filtering water entering and leaving the raceways and collecting surface swabs from walls and baffles. DNA was extracted and either the V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq or the V1 to V9 regions were amplified and sequenced on a PacBio Sequel II. During the course of this study the raceways walls were not cleaned after stocking the fish, so the age of the fish reflects the age of the biofilm. The data analysis revealed the biofilms in raceways harboring the youngest fish had a simple community and in raceways that harbored older fish had more complex communities. We also cultured hundreds of bacteria from this site. 570 isolates were grown up in broth, their DNA extracted and the V1-V9 region of the 16S rRNA gene amplified using the Shoreline Biome DNA V1-V9 kit. Six different extraction plates were used and six SMRTbells with different barcodes were used to create libraries that were sequenced on a PacBio Sequel II. The data was analyzed using the SBanalyzer, DADA2 and Qiime2. This allowed the identification of the isolates. We identified bacteria that belonged to 22 families and 48 genera. In summary, PacBio, full-length 16S rRNA gene sequences provides high resolution insights into microbial communities and allows the rapid identification of bacteria.

Learning Objectives:

1. Gain an understanding of the changes in composition and complexity of microbial biofilms over time

2. Learn a novel approach for identifying hundreds of bacterial strains by sequencing the full-length 16S rRNA gene using a PacBio Sequel2