Vibrio alginolyticus is a naturally occurring marine bacterium, recognized as an emerging pathogen in humans and animals and the second most common cause of vibriosis in the U.S. However, information regarding the physiology and growth of this species in the environment is limited. Here we evaluated the effects of temperature, salinity, and iron condition on the growth response across unique V. alginolyticus strains. A combination of growth kinetics and gas chromatography-mass spectrometry-based metabolomics studies was used to evaluate the optimal and tolerable ranges of growth and to characterize the metabolic effects of iron supplementation. All V. alginolyticus strains tested demonstrated broad temperature and salinity tolerance, resulting in growth at all measured temperatures (24°C–40°C) and salinities between 1% and 6% (wt/vol) NaCl with optimal growth between 30°C–36°C and 2%–4% NaCl. Environmental strains showed no growth limitation at iron concentrations ranging from 0.5- to 20.0-µM ferric Fe but demonstrated reduced growth at 0.2 µM. Likewise, the number of significantly upregulated metabolites in V. alginolyticus cultures grown in iron-replete (4-µM) media was greater than that in iron-deficient (~0 µM) media but varied with prior growth conditions. Detected compounds were associated with key metabolic pathways, namely, amino acid, carbohydrate, lipid, and nucleotide metabolism, suggesting that introduced iron facilitated broad activation of V. alginolyticus metabolism and helped to promote growth responses. Combined, these results demonstrate that V. alginolyticus strains are capable of rapid growth under a broad range of favorable temperature and salinity levels, which can be affected by the presence of iron. IMPORTANCE: Transmission of V. alginolyticus occurs opportunistically through direct seawater exposure and is a function of its abundance in the environment. Like other Vibrio spp., V. alginolyticus are considered conditionally rare taxa in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions, which remain poorly defined. Prior research has established the importance of temperature and salinity as the major determinants of Vibrio geographical and temporal range. However, bloom formation can be strongly influenced by other factors that may be more episodic and localized, such as changes in iron availability. Here we confirm the broad temperature and salinity tolerance of V. alginolyticus and demonstrate the importance of iron supplementation as a key factor for growth in the absence of thermal or osmotic stress. The results of this research highlight the importance of episodic iron input as a crucial metric to consider for the assessment of V. alginolyticus risk.

Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but pre-concentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for one year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280 µL) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69 to 0.82). To compensate for the method's high limit of detection (approximately 10⁶–10⁷ copies L⁻¹ in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100 000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

Prior research has suggested that the use of Aliivibrio fischeri -derived donor plasmids with the pES213 origin of replication may provide increased plasmid stability for the tagging of vibrios compared to Escherichia coli -derived p15A plasmids. Here, we present a structured protocol for conjugation-based tagging of vibrios using the pES213-derived plasmid pVSV102 and evaluate the plasmid stability of tagged strains.

Medical demands during the COVID-19 pandemic have triggered a grave shortage of medical-grade personal protective equipment (PPE), especially, N95 respirators. N95 respirators are critical for the personal protection of medical providers and others when being exposed to individuals with infections caused by the SARS-CoV-2 coronavirus. To address the shortage of N95 respirators, innovative methods are needed to decontaminate coronaviruses from N95 respirators, allowing them to be safely reused by healthcare workers. For this research, we use a commercial ozone disinfecting cabinet to examine the efficacy of ozone-based disinfection of a conservative surrogate virus for SARS-CoV-2, the MS2 bacteriophage. Treatment of mask materials with enhanced ozone treatment resulted in 2.38-log ₁₀ (>99%) reduction of phage from household dust masks and a range of 1.43-log ₁₀ (96.2%) to 4-log ₁₀ (99.99%) reductions of phage from common N95 mask materials.

The front line of pathogen defense in corals is the mucus membrane. This membrane coats the surface body wall creating a semi-impermeable layer that inhibits pathogen entry from the ambient water both physically and biologically through mutualistic antagonism from resident mucus microbes.

During the COVID-19 pandemic, the detection and sequencing of SARS-CoV-2 from wastewater proved to be a valuable tool in assessing trends at the community level. Several whole genome enrichment methods have been proposed for sequencing SARS-CoV-2 from the mixed wastewater community, but there is little consensus on the most appropriate sequencing methods for variant detection or abundance estimations. Few studies have elucidated the errors associated with these methods or have established minimum sequencing requirements for correct interpretation of the results. To address these needs, we systematically assessed the efficacy of three tiled amplicon enrichment methods (Freed/Midnight, ARTIC V4, NEB VarSkip) for whole genome sequencing of SARS-CoV-2 variants using mock wastewater communities with variants at known proportions. We found the ARTIC V4 approach yielded the most accurate results for variant identification and variant abundance estimation, followed by the NEB VarSkip approach. Conversely, the NEB VarSkip method obtained the highest genomic coverage, with the ARTIC V4 method achieving the second highest coverage. Finally, we determined that the Freed/Midnight library preparation methods are not well-suited for use with short read sequencing. Based on the present results, the ARTIC V4 workflow appears to be the most robust and cost-effective approach for monitoring circulating SARS-CoV-2 variants with wastewater surveillance. IMPORTANCE This work is informative for practitioners of wastewater-based epidemiology. Here, we detail a systematic comparison of three tiled amplicon sequencing approaches for enrichment of SARS-CoV-2 variants from wastewater. Using mock communities of known variant composition, we validate the analysis methods previously published by Baaijens et al. in Genome Biology (2022) for estimating variant abundance from wastewater using an RNAseq pipeline, kallisto. We provide recommendations for minimum sequencing requirements for accurate abundance estimates of SARS-CoV-2 variants in wastewater. The sequences generated from the mock communities have been uploaded to NCBI’s Sequence Read Archive and will be useful to other practitioners seeking to validate their sequencing methods or bioinformatic pipelines.