Highly sensitive wastewater surveillance of SARS-CoV-2 variants by targeted next-generation amplicon sequencing provides early warning of incursion in Victoria, Australia.

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread.IMPORTANCEThis study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method's flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

Antibiotics reduce bacterial load in Exaiptasia diaphana, but biofilms hinder its development as a gnotobiotic coral model.

Coral reefs are declining due to anthropogenic disturbances, including climate change. Therefore, improving our understanding of coral ecosystems is vital, and the influence of bacteria on coral health has attracted particular interest. However, a gnotobiotic coral model that could enhance studies of coral-bacteria interactions is absent. To address this gap, we tested the ability of treatment with seven antibiotics for 3 weeks to deplete bacteria in Exaiptasia diaphana, a sea anemone widely used as a coral model. Digital droplet PCR (ddPCR) targeting anemone Ef1-α and bacterial 16S rRNA genes was used to quantify bacterial load, which was found to decrease six-fold. However, metabarcoding of bacterial 16S rRNA genes showed that alpha and beta diversity of the anemone-associated bacterial communities increased significantly. Therefore, gnotobiotic E. diaphana with simplified, uniform bacterial communities were not generated, with biofilm formation in the culture vessels most likely impeding efforts to eliminate bacteria. Despite this outcome, our work will inform future efforts to create a much needed gnotobiotic coral model.

Exploring microbiome engineering as a strategy for improved thermal tolerance in Exaiptasia diaphana.

AIMS: Fourteen percent of all living coral, equivalent to more than all the coral on the Great Barrier Reef, has died in the past decade as a result of climate change-driven bleaching. Inspired by the 'oxidative stress theory of coral bleaching', we investigated whether a bacterial consortium designed to scavenge free radicals could integrate into the host microbiome and improve thermal tolerance of the coral model, Exaiptasia diaphana. METHODS AND RESULTS: E. diaphana anemones were inoculated with a consortium of high free radical scavenging (FRS) bacteria, a consortium of congeneric low FRS bacteria, or sterile seawater as a control, then exposed to elevated temperature. Increases in the relative abundance of Labrenzia during the first 2 weeks following the last inoculation provided evidence for temporary inoculum integration into the E. diaphana microbiome. Initial uptake of other consortium members was inconsistent, and these bacteria did not persist either in E. diaphana's microbiome over time. Given their non-integration into the host microbiome, the ability of the FRS consortium to mitigate thermal stress could not be assessed. Importantly, there were no physiological impacts (negative or positive) of the bacterial inoculations on the holobiont. CONCLUSIONS: The introduced bacteria were not maintained in the anemone microbiome over time, thus, their protective effect is unknown. Achieving long-term integration of bacteria into cnidarian microbiomes remains a research priority. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiome engineering strategies to mitigate coral bleaching may assist coral reefs in their persistence until climate change has been curbed. This study provides insights that will inform microbiome manipulation approaches in coral bleaching mitigation research.

Microbiota characterization of Exaiptasia diaphana from the Great Barrier Reef.

BACKGROUND: Coral reefs have sustained damage of increasing scale and frequency due to climate change, thereby intensifying the need to elucidate corals' biological characteristics, including their thermal tolerance and microbial symbioses. The sea anemone, Exaiptasia diaphana, has proven an ideal coral model for many studies due to its close phylogenetic relationship and shared traits, such as symbiosis with algae of the family Symbiodiniaceae. However, established E. diaphana clonal lines are not available in Australia thus limiting the ability of Australian scientists to conduct research with this model. To help address this, the bacterial and Symbiodiniaceae associates of four Great Barrier Reef (GBR)-sourced E. diaphana genotypes established in laboratory aquaria and designated AIMS1-4, and from proxies of wild GBR E. diaphana were identified by metabarcoding of the bacterial 16S rRNA gene and eukaryotic rRNA gene ITS2 region. The relationship between AIMS1-4 and their bacterial associates was investigated, as was bacterial community phenotypic potential. Existing data from two existing anemone clonal lines, CC7 and H2, were included for comparison. RESULTS: Overall, 2238 bacterial amplicon sequence variants (ASVs) were observed in the AIMS1-4 bacterial communities, which were dominated by Proteobacteria and Bacteroidetes, together comprising > 90% relative abundance. Although many low abundance bacterial taxa varied between the anemone genotypes, the AIMS1-4 communities did not differ significantly. A significant tank effect was identified, indicating an environmental effect on the microbial communities. Bacterial community richness was lower in all lab-maintained E. diaphana compared to the wild proxies, suggesting a reduction in bacterial diversity and community phenotypic potential due to culturing. Seventeen ASVs were common to every GBR lab-cultured anemone, however five were associated with the Artemia feedstock, making their specific association to E. diaphana uncertain. The dominant Symbiodiniaceae symbiont in all GBR anemones was Breviolum minutum. CONCLUSION: Despite differences in the presence and abundance of low abundance taxa, the bacterial communities of GBR-sourced lab-cultured E. diaphana are generally uniform and comparable to communities reported for other lab-cultured E. diaphana. The data presented here add to the global E. diaphana knowledge base and make an important contribution to the establishment of a GBR-sourced coral model organism.

The Effect of Thermal Stress on the Bacterial Microbiome of Exaiptasia diaphana.

Coral bleaching linked to climate change has generated interest in the response of coral's bacterial microbiome to thermal stress. The sea anemone, Exaiptasia diaphana, is a popular coral model, but the response of its bacteria to thermal stress has been barely explored. To address this, we compared the bacterial communities of Great Barrier Reef (GBR) E. diaphana maintained at 26 °C or exposed to increasing temperature (26-33 °C) over two weeks. Communities were analyzed by metabarcoding of the bacterial 16S rRNA gene. Bleaching and Symbiodiniaceae health were assessed by Symbiodiniaceae cell density and dark-adapted quantum yield (Fv/Fm), respectively. Significant bleaching and reductions in Fv/Fm occurred in the heat-treated anemones above 29 °C. Overall declines in bacterial alpha diversity in all anemones were also observed. Signs of bacterial change emerged above 31 °C. Some initial outcomes may have been influenced by relocation or starvation, but collectively, the bacterial community and taxa-level data suggested that heat was the primary driver of change above 32 °C. Six bacterial indicator species were identified as potential biomarkers for thermal stress. We conclude that the bacterial microbiome of GBR E. diaphana is generally stable until a thermal threshold is surpassed, after which significant changes occur.