Refining real-time predictions of Vibrio vulnificus concentrations in a tropical urban estuary by incorporating dissolved organic matter dynamics.

The south shore of O'ahu, Hawai'i is one of the most visited coastal tourism areas in the United States with some of the highest instances of recreational waterborne disease. A population of the pathogenic bacterium Vibrio vulnificus lives in the estuarine Ala Wai Canal in Honolulu which surrounds the heavily populated tourism center of Waikiki. We developed a statistical model to predict V. vulnificus dynamics in this system using environmental measurements from moored oceanographic and atmospheric sensors in real time. During a year-long investigation, we analyzed water from 9 sampling events at 3 depths and 8 sites along the canal (n = 213) for 36 biogeochemical variables and V. vulnificus concentration using quantitative polymerase chain reaction (qPCR) of the hemolysin A gene (vvhA). The best multiple linear regression model of V. vulnificus concentration, explaining 80% of variation, included only six predictors: 5-day average rainfall preceding water sampling, daily maximum air temperature, water temperature, nitrate plus nitrite, and two metrics of humic dissolved organic matter (DOM). We show how real-time predictions of V. vulnificus concentration can be made using these models applied to the time series of water quality measurements from the Pacific Islands Ocean Observing System (PacIOOS) as well as the PacIOOS plume model based on the Waikiki Regional Ocean Modeling System (ROMS) products. These applications highlight the importance of including DOM variables in predictive modeling of V. vulnificus and the influence of rain events in elevating nearshore concentrations of V. vulnificus. Long-term climate model projections of locally downscaled monthly rainfall and air temperature were used to predict an overall increase in V. vulnificus concentration of approximately 2- to 3-fold by 2100. Improving these predictive models of microbial populations is critical for management of waterborne pathogen risk exposure, particularly in the wake of a changing global climate.

Variable Freshwater Influences on the Abundance of Vibrio vulnificus in a Tropical Urban Estuary.

To better understand the controls on the opportunistic human pathogen Vibrio vulnificus in warm tropical waters, we conducted a year-long investigation in the Ala Wai Canal, a channelized estuary in Honolulu, HI. The abundance of V. vulnificus, as determined by quantitative PCR (qPCR) of the hemolysin gene (vvhA), varied spatially and temporally by nearly 4 orders of magnitude (≤3 to 14,000 mL-1). Unlike in temperate and subtropical systems, temperatures were persistently warm (19 to 31°C) and explained little of the variability in V. vulnificus abundance. Salinity (1 to 36 ppt) had a significant, but nonlinear, relationship with V. vulnificus abundance with the highest vvhA concentrations (>2,500 mL-1) observed only at salinities from 7 to 22 ppt. V. vulnificus abundances were lower on average during the summer dry season, when waters were warmer but more saline. The highest canal-wide average abundances were observed during a time of modest rainfall, when moderate salinities and elevated concentrations of reduced nitrogen species and silica suggested a groundwater influence. Parallel quantification of the vcgC gene suggested that C-type strains, which are responsible for most human infections, comprised 25% of the total V. vulnificus on average, but their relative contribution was greater at higher salinities, suggesting a broader salinity tolerance. Generalized regression models suggested that up to 67% of sample-to-sample variation (n = 202) in log-transformed V. vulnificus abundance was explained using the measured environmental variables, and up to 97% of the monthly variation in canal-wide average concentrations (n = 13) was explained with the best subset of four variables. IMPORTANCE Our data illustrate that, in the absence of strong seasonal variation in water temperature in the tropics, variation in salinity driven by rainfall becomes a primary controlling variable on V. vulnificus abundance. There is thus a tendency for a rainfall-driven seasonal cycle in V. vulnificus abundance which is inverted from the temperature-driven seasonal cycle at higher latitudes. However, stochasticity in rainfall and its nonlinear, indirect effects on V. vulnificus concentration means that high abundances can occur at any location in the canal at any time of year, making it challenging to predict concentrations of this pathogen at a high temporal or spatial resolution. Much of the variability in canal-wide average concentrations, on the other hand, was explained by a few variables that reflect the magnitude of freshwater input to the system, suggesting that relative risk of exposure to this pathogen could be predicted as an average for the system.

Sulfur cycling and host-virus interactions in Aquificales-dominated biofilms from Yellowstone's hottest ecosystems.

Modern linkages among magmatic, geochemical, and geobiological processes provide clues about the importance of thermophiles in the origin of biogeochemical cycles. The aim of this study was to identify the primary chemoautotrophs and host-virus interactions involved in microbial colonization and biogeochemical cycling at sublacustrine, vapor-dominated vents that represent the hottest measured ecosystems in Yellowstone National Park (~140 °C). Filamentous microbial communities exposed to extreme thermal and geochemical gradients were sampled using a remotely operated vehicle and subjected to random metagenome sequencing and microscopic analyses. Sulfurihydrogenibium (phylum Aquificae) was the predominant lineage (up to 84% relative abundance) detected at vents that discharged high levels of dissolved H2, H2S, and CO2. Metabolic analyses indicated carbon fixation by Sulfurihydrogenibium spp. was powered by the oxidation of reduced sulfur and H2, which provides organic carbon for heterotrophic community members. Highly variable Sulfurihydrogenibium genomes suggested the importance of intra-population diversity under extreme environmental and viral pressures. Numerous lytic viruses (primarily unclassified taxa) were associated with diverse archaea and bacteria in the vent community. Five circular dsDNA uncultivated virus genomes (UViGs) of ~40 kbp length were linked to the Sulfurihydrogenibium metagenome-assembled genome (MAG) by CRISPR spacer matches. Four UViGs contained consistent genome architecture and formed a monophyletic cluster with the recently proposed Pyrovirus genus within the Caudovirales. Sulfurihydrogenibium spp. also contained CRISPR arrays linked to plasmid DNA with genes for a novel type IV filament system and a highly expressed ß-barrel porin. A diverse suite of transcribed secretion systems was consistent with direct microscopic analyses, which revealed an extensive extracellular matrix likely critical to community structure and function. We hypothesize these attributes are fundamental to the establishment and survival of microbial communities in highly turbulent, extreme-gradient environments.

CoCoNet: an efficient deep learning tool for viral metagenome binning.

MOTIVATION: Metagenomic approaches hold the potential to characterize microbial communities and unravel the intricate link between the microbiome and biological processes. Assembly is one of the most critical steps in metagenomics experiments. It consists of transforming overlapping DNA sequencing reads into sufficiently accurate representations of the community's genomes. This process is computationally difficult and commonly results in genomes fragmented across many contigs. Computational binning methods are used to mitigate fragmentation by partitioning contigs based on their sequence composition, abundance or chromosome organization into bins representing the community's genomes. Existing binning methods have been principally tuned for bacterial genomes and do not perform favorably on viral metagenomes. RESULTS: We propose Composition and Coverage Network (CoCoNet), a new binning method for viral metagenomes that leverages the flexibility and the effectiveness of deep learning to model the co-occurrence of contigs belonging to the same viral genome and provide a rigorous framework for binning viral contigs. Our results show that CoCoNet substantially outperforms existing binning methods on viral datasets. AVAILABILITY AND IMPLEMENTATION: CoCoNet was implemented in Python and is available for download on PyPi (https://pypi.org/). The source code is hosted on GitHub at https://github.com/Puumanamana/CoCoNet and the documentation is available at https://coconet.readthedocs.io/en/latest/index.html. CoCoNet does not require extensive resources to run. For example, binning 100k contigs took about 4 h on 10 Intel CPU Cores (2.4 GHz), with a memory peak at 27 GB (see Supplementary Fig. S9). To process a large dataset, CoCoNet may need to be run on a high RAM capacity server. Such servers are typically available in high-performance or cloud computing settings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research.

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai'i at Manoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai'i. Due to its high ecological diversity across tractable areas of land and sea, Hawai'i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Viruses in the Oceanic Basement.

Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 105 to 2 × 105 ml-1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome.IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not well understood. Viruses in particular, although integral to the origins, evolution, and ecology of all life on earth, have never been documented in basement fluids. This report provides the first estimate of free virus particles (virions) within fluids circulating through the extrusive basalt of the seafloor and describes the morphological and genetic signatures of basement viruses. These data push the known geographical limits of the virosphere deep into the ocean basement and point to a wealth of novel viral diversity, exploration of which could shed light on the early evolution of viruses.

Differential specificity of selective culture media for enumeration of pathogenic vibrios: advantages and limitations of multi-plating methods.

Plating environmental samples on vibrio-selective chromogenic media is a commonly used technique that allows one to quickly estimate concentrations of putative vibrio pathogens or to isolate them for further study. Although this approach is convenient, its usefulness depends directly on how well the procedure selects against false positives. We tested whether a chromogenic medium, CHROMagar Vibrio (CaV), used alone (single-plating) or in combination (double-plating) with a traditional medium thiosulfate-citrate-bile-salts (TCBS), could improve the discrimination among three pathogenic vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) and thereby decrease the number of false-positive colonies that must be screened by molecular methods. Assays were conducted on water samples from two estuarine environments (one subtropical, one tropical) in a variety of seasonal conditions. The results of the double-plating method were confirmed by PCR and 16S rRNA sequencing. Our data indicate that there is no significant difference in the false-positive rate between CaV and TCBS when using a single-plating technique, but determining color changes on the two media sequentially (double-plating) reduced the rate of false positive identification in most cases. The improvement achieved was about two-fold on average, but varied greatly (from 0- to 5-fold) and depended on the sampling time and location. The double-plating method was most effective for V. vulnificus in warm months, when overall V. vulnificus abundance is high (false positive rates as low as 2%, n=178). Similar results were obtained for V. cholerae (minimum false positive rate of 16%, n=146). In contrast, the false positive rate for V. parahaemolyticus was always high (minimum of 59%, n=109). Sequence analysis of false-positive isolates indicated that the majority of confounding isolates are from the Vibrionaceae family, however, members of distantly related bacterial groups were also able to grow on vibrio-selective media, even when using the double-plating method. In conclusion, the double-plating assay is a simple means to increase the efficiency of identifying pathogenic vibrios in aquatic environments and to reduce the number of molecular assays required for identity confirmation. However, the high spatial and temporal variability in the performance of the media mean that molecular approaches are still essential to obtain the most accurate vibrio abundance estimates from environmental samples.

Draft genome sequence of marine alphaproteobacterial strain HIMB11, the first cultivated representative of a unique lineage within the Roseobacter clade possessing an unusually small genome.

Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.

Complete genome sequence of bacteriophage VvAW1, which infects Vibrio vulnificus.

Investigating the bacteriophages of vibrios has led to significant insights into the evolution and pathogenicity of their host strains. This report presents the first complete genome sequence of a bacteriophage that infects the deadly human pathogen Vibrio vulnificus. The phage was isolated from the surface waters of the Ala Wai Canal, which is part of an urban watershed in eastern Honolulu, Hawaii, USA. The phage particle is icosahedral, with a diameter of 35-40 nm, and a small non-contractile tail. The genome was sequenced in its entirety, rendering a 38 kb sequence located on a single, linear, circularly permuted chromosome. Here, we present the annotation and genomic features of the bacteriophage, VvAW1.

Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita.

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.