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Objectives: Rapid evolution of SARS-CoV-2 has resulted in the emergence of numerous variants, posing significant challenges to public health surveillance. Clinical genome sequencing, while valuable, has limitations in capturing the full epidemiological dynamics of circulating variants in the general population. This study aimed to monitor the SARS-CoV-2 variant community dynamics and evolution using receptor-binding domain (RBD) amplicon sequencing of wastewater samples. Methods: We sequenced wastewater from El Paso, Texas, over 17 months, compared the sequencing data with clinical genome data, and performed biodiversity analysis to reveal SARS-CoV-2 variant dynamics and evolution. Results: We identified 91 variants and observed waves of dominant variants transitioning from BA.2 to BA.2.12.1, BA.4&5, BQ.1, and XBB.1.5. Comparison with clinical genome sequencing data revealed earlier detection of variants and identification of unreported outbreaks. Our results also showed strong consistency with clinical data for dominant variants at the local, state, and national levels. Alpha diversity analyses revealed significant seasonal variations, with the highest diversity observed in winter. By segmenting the outbreak into lag, growth, stationary, and decline phases, we found higher variant diversity during the lag phase, likely due to lower inter-variant competition preceding outbreak growth. Conclusions: Our findings underscore the importance of low transmission periods in facilitating rapid mutation and variant evolution. Our approach, integrating RBD amplicon sequencing with wastewater surveillance, demonstrates effectiveness in tracking viral evolution and understanding variant emergence, thus enhancing public health preparedness. 

ABSTRACT Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, anotherVibriospp. gDNA, and an oligomer of 61–80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCEBacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of aVibrio hyugaensisbiofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.