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Vibrio spp. thrive in warm water and moderate salinity, and they are associated with aquatic invertebrates, notably crustaceans and zooplankton. At least twelve Vibrio spp. are known to cause infection in humans, and Vibrio cholerae is well documented as the etiological agent of pandemic cholera. Pathogenic non-cholera Vibrio spp., e.g., Vibrio parahaemolyticus and Vibrio vulnificus, cause gastroenteritis, septicemia, and other extra-intestinal infections. Incidence of vibriosis is rising globally, with evidence that anthropogenic factors, primarily emissions of carbon dioxide associated with atmospheric warming and more frequent and intense heatwaves, significantly influence environmental parameters, e.g., temperature, salinity, and nutrients, all of which can enhance growth of Vibrio spp. in aquatic ecosystems. It is not possible to eliminate Vibrio spp., as they are autochthonous to the aquatic environment and many play a critical role in carbon and nitrogen cycling. Risk prediction models provide an early warning that is essential for safeguarding public health. This is especially important for regions of the world vulnerable to infrastructure instability, including lack of 'water, sanitation, and hygiene' (WASH), and a less resilient infrastructure that is vulnerable to natural calamity, e.g., hurricanes, floods, and earthquakes, and/or social disruption and civil unrest, arising from war, coups, political crisis, and economic recession. Incorporating environmental, social, and behavioral parameters into such models allows improved prediction, particularly of cholera epidemics. We have reported that damage to WASH infrastructure, coupled with elevated air temperatures and followed by above average rainfall, promotes exposure of a population to contaminated water and increases the risk of an outbreak of cholera. Interestingly, global predictive risk models successful for cholera have the potential, with modification, to predict diseases caused by other clinically relevant Vibrio spp. In the research reported here, the focus was on environmental parameters associated with incidence and distribution of clinically relevant Vibrio spp. and their role in disease transmission. In addition, molecular methods designed for detection and enumeration proved useful for predictive modeling and are described, namely in the context of prediction of environmental conditions favorable to Vibrio spp., hence human health risk. This article is protected by copyright. All rights reserved.

The microbiological content of drinking water traditionally is determined by employing culture-dependent methods that are unable to detect all microorganisms, especially those that are not culturable. High-throughput sequencing now makes it possible to determine the microbiome of drinking water. Thus, the natural microbiota of water and water distribution systems can now be determined more accurately and analyzed in significantly greater detail, providing comprehensive understanding of the microbial community of drinking water applicable to public health. In this study, shotgun metagenomic analysis was performed to determine the microbiological content of drinking water and to provide a preliminary assessment of tap, drinking fountain, sparkling natural mineral, and non-mineral bottled water. Predominant bacterial species detected were members of the phyla Actinobacteria and Proteobacteria, notably the genera Alishewanella, Salmonella, and Propionibacterium in non-carbonated non-mineral bottled water, Methyloversatilis and Methylibium in sparkling natural mineral water, and Mycobacterium and Afipia in tap and drinking fountain water. Fecal indicator bacteria, i.e., Escherichia coli or enterococci, were not detected in any samples examined in this study. Bacteriophages and DNA encoding a few virulence-associated factors were detected but determined to be present only at low abundance. Antibiotic resistance markers were detected only at abundance values below our threshold of confidence. DNA of opportunistic plant and animal pathogens was identified in some samples and these included bacteria (Mycobacterium spp.), protozoa (Acanthamoeba mauritaniensis and Acanthamoeba palestinensis), and fungi (Melampsora pinitorqua and Chryosporium queenslandicum). Archaeal DNA (Candidatus Nitrosoarchaeum) was detected only in sparkling natural mineral water. This preliminary study reports the complete microbiome (bacteria, viruses, fungi, and protists) of selected types of drinking water employing whole-genome high-throughput sequencing and bioinformatics. Investigation into activity and function of the organisms detected is in progress.

Microorganisms are ubiquitous in the biosphere, playing a crucial role in both biogeochemistry of the planet and human health. However, identifying these microorganisms and defining their function are challenging. Widely used approaches in comparative metagenomics, 16S amplicon sequencing and whole genome shotgun sequencing (WGS), have provided access to DNA sequencing analysis to identify microorganisms and evaluate diversity and abundance in various environments. However, advances in parallel high-throughput DNA sequencing in the past decade have introduced major hurdles, namely standardization of methods, data storage, reproducible interoperability of results, and data sharing. The National Ecological Observatory Network (NEON), established by the National Science Foundation, enables all researchers to address queries on a regional to continental scale around a variety of environmental challenges and provide high-quality, integrated, and standardized data from field sites across the U.S. As the amount of metagenomic data continues to grow, standardized procedures that allow results across projects to be assessed and compared is becoming increasingly important in the field of metagenomics. We demonstrate the feasibility of using publicly available NEON soil metagenomic sequencing datasets in combination with open access Metagenomics Rapid Annotation using the Subsystem Technology (MG-RAST) server to illustrate advantages of WGS compared to 16S amplicon sequencing. Four WGS and four 16S amplicon sequence datasets, from surface soil samples prepared by NEON investigators, were selected for comparison, using standardized protocols collected at the same locations in Colorado between April-July 2014. The dominant bacterial phyla detected across samples agreed between sequencing methodologies. However, WGS yielded greater microbial resolution, increased accuracy, and allowed identification of more genera of bacteria, archaea, viruses, and eukaryota, and putative functional genes that would have gone undetected using 16S amplicon sequencing. NEON open data will be useful for future studies characterizing and quantifying complex ecological processes associated with changing aquatic and terrestrial ecosystems.

Background The global pandemic of Coronavirus infectious disease 2019 (COVID-19), caused by SARS-CoV-2, has plunged the world into both social and economic disarray, with vaccines still emerging and a continued paucity of personal protective equipment; the pandemic has also highlighted the potential for rapid emergence of aggressive respiratory pathogens and the need for preparedness. Avian immunoglobulins (IgY) have been previously shown in animal models to protect against new infection and mitigate established infection when applied intranasally. We carried out a proof-of-concept study to address the feasibility of using such antibodies as mucosally-applied prophylaxis against SARS-CoV-2. Methods Hens were immunized with recombinant S1 spike glycoprotein of the virus, and the resulting IgY was evaluated for binding specificity, inhibition of glycoprotein binding to angiotensin converting enzyme-2 (ACE2) protein (the requisite binding site for the virus), and inhibition of viral replication in Vero cell culture. Results Titers of anti-S1 glycoprotein IgY were evident in yolks at 14 days post-immunization, peaking at 21 days, and at peak concentrations of 16.8 mg/ml. IgY showed strong and significant inhibition of S1/ACE2 binding interactions, and significantly inhibited viral replication at a concentration of 16.8 mg/ml. Four weeks’ collection from eggs of two hens produced a total of 1.55 grams of IgY. Conclusions In this proof-of-concept study we showed that avian immunoglobulins (IgY) raised against a key virulence factor of the SARS-CoV-2 virus successfully inhibited the critical initial adhesion of viral spike glycoproteins to human ACE2 protein receptors and inhibited viral replication in vitro, in a short period using only two laying hens. We conclude that production of large amounts of IgY inhibiting viral binding and replication of SARS-CoV-2 is feasible, and that incorporation of this or similar material into an intranasal spray and/or other mucosal protecting products may be effective at reducing infection and spread of COVID-19.

Cholera is a severe diarrheal disease caused by the consumption of food or water contaminated with the aquatic gram-negative bacterium Vibrio cholerae. Infected hosts will experience vomiting and severe watery diarrhea and if not treated properly will ultimately succumb to death by dehydration. Due to the global prevalence and severity of cholera, V. cholerae has been extensively studied in both environmental and laboratory settings. Herein, we describe proper V. cholerae maintenance, in addition to classical and El Tor biotype culturing in a laboratory setting.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000's clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.