Probiotic Solutions to Improve Pacific Oyster Larvae Growth and Development
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AGRICULTURAL RESEARCH FOUNDATION INTERIM REPORT FUNDING CYCLE 2020 – 2022 TITLE: Probiotic solutions to improve Pacific oyster larvae growth and development RESEARCH LEADER: Ryan Mueller (PI, OSU), Carla Schubiger (CoI, OSU), Chris Langdon (CoI, OSU), MK English (Grad. Student, OSU) COOPERATORS: Sandra Loesgen (U. of Florida), Annika Jagels (Postdoc, U. of Florida) EXECUTIVE SUMMARY: The work completed during the first year of this ARF project was focused on identifying and characterizing bacterial isolates that can be used as probiotics in oyster aquaculture by improving the growth and health of oyster larvae and juveniles and increasing their resistance to disease. The genetic and physiological mechanisms of probiotics that underlie these outcomes in oysters are currently poorly defined. Therefore, the primary goal of our research was to define the antagonistic mechanisms of several isolated oyster probiotics by performing co-culture experiments between probiotics and a known oyster pathogen (Vibrio coralliilyticus strain RE22) and by performing bioinformatic analyses of genome sequences of the probiotics. For the latter, genome sequences of probiotics were compared to sequences of known proteins and genes to identify potential mechanisms of antagonism between probiotics and pathogens. DNA sequences with matches to factors known to be involved in antagonisms will be targeted in the analysis of data from subsequent transcriptomic experiments of co-cultures of probiotics and pathogens with and without oyster larvae. Due to the challenges of performing in-person laboratory research during the COVID-19 pandemic, our focus over the first year of this project turned to remotely performing detailed bioinformatic analyses to discover the genes and gene products that are possibly responsible for killing or inhibitory activity of probiotic strains against the oyster bacterial pathogen, V. coralliilyticus strain RE22. Our analyses have led us to focus on one probiotic strain in particular, which we have identified as Epibacterium mobile strain B11. Results of comparative genomic analyses confidently mark this bacterium as a close phylogenetic relative of other known antibiotic-producing strains of bacteria. Additional bioinformatic analyses have shown that the E. mobile strain B11 genome encodes genes for the production of a relatively novel antibiotic, Tropodithietic Acid (TDA), which is also produced by a well-studied probiotic of shellfish and finfish, Phaeobacter inhibens (Zhao et al., 2019). Additionally, we have obtained preliminary confirmation of growth-stage specific production of TDA by strain B11 with mass spectrometry. Intriguingly, analysis of the genome of E. mobile strain B11 suggests that, in addition to the production of TDA, this bacterium may produce entirely novel secondary metabolites that may have antagonistic activity against other bacteria. We are currently performing experiments to confirm the production of these new molecules and to explore their killing and/or inhibitory activity against other bacteria. Although in-person experimental work has been slowed due to the pandemic, we have completed several preliminary experiments to define the parameters of our proposed co-culture experiments. We are currently in the process of setting up these co-culture experiments between the probiotic strains DM14 and B11 to determine the genes expressed in the presence of V. coralliilyticus strain RE22, and with the goal of providing further confirmation of the role of these genes in secondary metabolite production and to potentially identify additional genetic mechanisms responsible for probiotic activity in these strains. OBJECTIVES: The primary objective of our work is to investigate the interactions between probiotics, pathogens, and oysters, in order to better define how probiotics enhance growth and suppress disease during larval and juvenile oyster development. 1 PROCEDURES: New Probiotic Isolation and Testing Probiotics bacterial strains used in this project were isolated from various sources using a uniform screening method. The DM14 probiotic isolate, which was the primary isolate identified for use in the original ARF proposal, was isolated from oyster spat that had survived a mortality event at Hatfield, with the assumption that the bacteria associated with this spat may have played a protective role against disease. This spat was crushed and plated on agar plates containing LB plus Seawater to isolate bacteria capable of growth on this medium. After overnight incubation at 25 °C, a random colony was picked, cultured in LB plus seawater, and stocked with glycerol at -80 °C. The second probiotic strain, B11, which we have subsequently focused on in our research this year (see reasons below), was isolated directly from oyster tissue. This tissue was dissected from the animal and heated in a microcentrifuge tube for 10 min in a 100 °C water bath, and plated on an agar plate containing LB plus 3% NaCl (LBS). After overnight incubation at 25 °C, an off-white colony was picked, cultured in LBS, and stocked with glycerol at -80 °C. Both of these probiotic strains, along with all other putative probiotic isolates used in our project were screened for antagonistic activity against the oyster pathogen, V. coralliilyticus strain RE22, using a uniform assay. Frozen stocks of the probiotics and V. coralliilyticus strain RE22 were struck on LBS plates and grown overnight at 25 °C. Single colonies were grown overnight in 5 ml LBS and shaking at 25 °C. The V. coralliilyticus strain RE22 culture was diluted to approximately 1E05 cfu/ml with LBS, and 50 µl spread on LBS plates with glass beads. After slightly drying the plates, 10 µl of probiotic culture was spotted onto the RE22 lawn, and the plate incubated overnight at 25 °C. Zones of inhibitions in the RE22 lawn indicated probiotic activity. 16S Sequencing and Strain Identification We sequenced the 16S rRNA gene of each isolate to rule out the possibility that one or more were duplicates and to confirm their taxonomic identity. A phenol:chloroform DNA extraction method was used to isolate DNA from 1 ml of overnight cultures of each probiotic. We then ran PCR to amplify the 16S rRNA gene using the primer pair 8F/1513R, creating a ~1500 bp gene product (Figure 1). DNA was cleaned and then sent to the CGRB for Sanger sequencing. Sequencing generated a forward and reverse read for each sample. We trimmed low-quality ends from these reads determined by a base call intensity chromatogram, then overlaid the trimmed forward and reverse reads in the Geneious Prime application to create a consensus sequence. Some reverse reads were consistently low quality, so for those samples, only the forward read was used. Finally, we analyzed each consensus sequence with the BLAST software to identify the best matches to a database of all known prokaryotic 16S Ribosomal RNA Sequences. Best matches to each probiotic 16S sequence are reported in Table 1. If there was more than one close match based on these parameters, the isolate was only identified to the genus level rather than species. Antagonism Assays We initially selected DM14 as the probiotic to be used in our co-culture experiment based on its fast, robust growth compared to the other strains. We performed a series of preliminary experiments to prepare for the main co-culture experiment, with the goal of identifying the time point where the ratio between probiotic to pathogen cells was greatest in order to sample for transcriptome analysis. To accurately estimate cell counts to use in experiments, we first established the relationship between optical density of cultures and cells per ml, which was used to identify when a shift in relative abundance occurred between the pathogen and probiotic. Prior to creating a co-culture, we first established equations to get a rough estimate of the relationship between OD600 (optical density at 600 nm, a common way to measure cell culture density) and cell density in CFU/ml (colony-forming units per ml). DM14 and RE22 were cultured separately overnight. A series of dilutions was used to provide a range of OD600 readings. These dilutions were then further diluted to a concentration low enough to be counted using a plating method. We calculated an 2 equation for each strain relating CFU counts with OD600 readings for each culture (R values of 0.44 and 2 0.41 for DM14 and RE22, respectively; Figure 2). This relationship was used in these preliminary experiments, but we ultimately decided to use a Guava flow cytometer for more accurate cell measurements in the main experiment. The first preliminary co-culture spanned a 24-hour incubation period with sampling at 1, 2, 4, 12, and 24 hours. Briefly, cells from overnight cultures of DM14 and RE22 washed with ASW. Using the established equations to relate OD600 to CFU/ml, the probiotic DM14 was stocked at a density of 6E04 CFU/ml and the pathogen RE22 at 3E04 CFU/ml in the co-culture. We used the differential medium TCBS agar, which selectively grows vibrios (RE22), to subtract out the RE22 counts from the combined counts on ZMB plates, which grows both indiscriminately. RE22 appeared to decrease in abundance around 4 hours, so we chose to shorten co-culture incubation times for the next preliminary experiment. The second preliminary co-culture was a five-hour incubation. Aliquots were removed from the culture at 1 hour intervals. These aliquots were then diluted to give a countable