Examining the immunotoxicity of oil sands process affected waters using a human macrophage cell line.

Oil sands process affected water (OSPW) is produced during the surface mining of the oil sands bitumen deposits in Northern Alberta. OSPW contains variable quantities of organic and inorganic components causing toxic effects on living organisms. Advanced Oxidation Processes (AOPs) are widely used to degrade toxic organic components from OSPW including naphthenic acids (NAs). However, there is no established biological procedure to assess the effectiveness of the remediation processes. Our previous study showed that human macrophage cells (THP-1) can be used as a bioindicator system to evaluate the effectiveness of OSPW treatments through examining the proinflammatory gene transcription levels. In the present study, we investigated the immunotoxicological changes in THP-1 cells following exposure to untreated and AOP-treated OSPW. Specifically, using proinflammatory cytokine protein secretion assays we showed that AOP treatment significantly abrogates the ability of OSPW to induce the secretion of IL-1ß, IL-6, IL-8, TNF-α, IL-1Ra and MCP-1. By measuring transcriptional activity as well as surface protein expression levels, we also showed that two select immune cell surface markers, CD40 and CD54, were significantly elevated following OSPW exposure. However, AOP treatments abolished the immunostimulatory properties of OSPW to enhance the surface expression of these immune proteins. Finally, a transcriptome-based approach was used to examine the proinflammatory effects of OSPW as well as the abrogation of immunotoxicity following AOP treatments. Overall, this research shows how a human macrophage cell-based biomonitoring system serves as an effective in vitro tool to study the immunotoxicity of OSPW samples before and after targeted remediation strategies.

Using immune cell-based bioactivity assays to compare the inflammatory activities of oil sands process-affected waters from a pilot scale demonstration pit lake.

In this study, we provide evidence that oil sands process-affected waters (OSPW) contain factors that activate the antimicrobial and proinflammatory responses of immune cells. Specifically, using the murine macrophage RAW 264.7 cell line, we establish the bioactivity of two different OSPW samples and their isolated fractions. Here, we directly compared the bioactivity of two pilot scale demonstration pit lake (DPL) water samples, which included expressed water from treated tailings (termed the before water capping sample; BWC) as well as an after water capping (AWC) sample consisting of a mixture of expressed water, precipitation, upland runoff, coagulated OSPW and added freshwater. Significant inflammatory (i.e. macrophage activating) bioactivity was associated with the AWC sample and its organic fraction (OF), whereas the BWC sample had reduced bioactivity that was primarily associated with its inorganic fraction (IF). Overall, these results indicate that at non-toxic exposure doses, the RAW 264.7 cell line serves as an acute, sensitive and reliable biosensor for the screening of inflammatory constituents within and among discrete OSPW samples.

Abiotic and biotic constituents of oil sands process-affected waters.

The oil sands in Northern Alberta are the largest oil sands in the world, providing an important economic resource for the Canadian energy industry. The extraction of petroleum in the oil sands begins with the addition of hot water to the bituminous sediment, generating oil sands process-affected water (OSPW), which is acutely toxic to organisms. Trillions of litres of OSPW are stored on oil sands mining leased sites in man-made reservoirs called tailings ponds. As the volume of OSPW increases, concerns arise regarding the reclamation and eventual release of this water back into the environment. OSPW is composed of a complex and heterogeneous mix of components that vary based on factors such as company extraction techniques, age of the water, location, and bitumen ore quality. Therefore, the effective remediation of OSPW requires the consideration of abiotic and biotic constituents within it to understand short and long term effects of treatments used. This review summarizes selected chemicals and organisms in these waters and their interactions to provide a holistic perspective on the physiochemical and microbial dynamics underpinning OSPW .

Genomes of Vibrio metoecus co-isolated with Vibrio cholerae extend our understanding of differences between these closely related species.

BACKGROUND: Vibrio cholerae, the causative agent of cholera, is a well-studied species, whereas Vibrio metoecus is a recently described close relative that is also associated with human infections. The availability of V. metoecus genomes provides further insight into its genetic differences from V. cholerae. Additionally, both species have been co-isolated from a cholera-free brackish coastal pond and have been suggested to interact with each other by horizontal gene transfer (HGT). RESULTS: The genomes of 17 strains from each species were sequenced. All strains share a large core genome (2675 gene families) and very few genes are unique to each species (< 3% of the pan-genome of both species). This led to the identification of potential molecular markers-for nitrite reduction, as well as peptidase and rhodanese activities-to further distinguish V. metoecus from V. cholerae. Interspecies HGT events were inferred in 21% of the core genes and 45% of the accessory genes. A directional bias in gene transfer events was found in the core genome, where V. metoecus was a recipient of three times (75%) more genes from V. cholerae than it was a donor (25%). CONCLUSION: V. metoecus was misclassified as an atypical variant of V. cholerae due to their resemblance in a majority of biochemical characteristics. More distinguishing phenotypic assays can be developed based on the discovery of potential gene markers to avoid any future misclassifications. Furthermore, differences in relative abundance or seasonality were observed between the species and could contribute to the bias in directionality of HGT.

Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction.

Cholera is a severe diarrheal disease caused by Vibrio cholerae, a natural inhabitant of brackish water. Effective control of cholera outbreaks depends on prompt detection of the pathogen from clinical specimens and tracking its source in the environment. Although the epidemiology of cholera is well studied, rapid detection of V. cholerae remains a challenge, and data on its abundance in environmental sources are limited. Here, we describe a sensitive molecular quantification assay by qPCR, which can be used on-site in low-resource settings on water without the need for DNA extraction. This newly optimized method exhibited 100% specificity for total V. cholerae as well as V. cholerae O1 and allowed detection of as few as three target CFU per reaction. The limit of detection is as low as 5 × 103 CFU/L of water after concentrating biomass from the sample. The ability to perform qPCR on water samples without DNA extraction, portable features of the equipment, stability of the reagents at 4 °C and user-friendly online software facilitate fast quantitative analysis of V. cholerae. These characteristics make this assay extremely useful for field research in resource-poor settings and could support continuous monitoring in cholera-endemic areas.

Modular Molecular Weaponry Plays a Key Role in Competition Within an Environmental Vibrio cholerae Population.

The type VI secretion system (T6SS) operons of Vibrio cholerae contain extraordinarily diverse arrays of toxic effector and cognate immunity genes, which are thought to play an important role in the environmental lifestyle and adaptation of this human pathogen. Through the T6SS, proteinaceous "spears" tipped with antibacterial effectors are injected into adjacent cells, killing those not possessing immunity proteins to these effectors. Here, we investigate the T6SS-mediated dynamics of bacterial competition within a single environmental population of V. cholerae. We show that numerous members of a North American V. cholerae population possess strain-specific repertoires of cytotoxic T6SS effector and immunity genes. Using pairwise competition assays, we demonstrate that the vast majority of T6SS-mediated duels end in stalemates between strains with different T6SS repertoires. However, horizontally acquired effector and immunity genes can significantly alter the outcome of these competitions. Frequently observed horizontal gene transfer events can both increase or reduce competition between distantly related strains by homogenizing or diversifying the T6SS repertoire. Our results also suggest temperature-dependent outcomes in T6SS competition, with environmental isolates faring better against a pathogenic strain under native conditions than under those resembling a host-associated environment. Taken altogether, these interactions produce density-dependent fitness effects and a constant T6SS-mediated arms race in individual V. cholerae populations, which could ultimately preserve intraspecies diversity. Since T6SSs are widespread, we expect within-population diversity in T6SS repertoires and the resulting competitive dynamics to be a common theme in bacterial species harboring this machinery.

Population Analysis of Vibrio cholerae in Aquatic Reservoirs Reveals a Novel Sister Species (Vibrio paracholerae sp. nov.) with a History of Association with Humans.

Most efforts to understand the biology of Vibrio cholerae have focused on a single group, the pandemic-generating lineage harboring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in the V. cholerae populations inhabiting regions with a history of cholera cases and those lacking such a history, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka, Bangladesh (where cholera is endemic), located in the Ganges Delta, and those in Falmouth, MA (no known history of cholera), a small coastal town on the United States east coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka, which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny for the V. cholerae species and were sufficiently differentiated genetically and phenotypically to form a novel species. A retrospective search revealed that strains from this species have been anecdotally found from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhea. In 1935, Gardner and Venkatraman unofficially referred to a member of this group as Vibrio paracholerae. In recognition of this earlier designation, we propose the name Vibrio paracholerae sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species. IMPORTANCE Cholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution, and environmental adaptation of the causative agent (Vibrio cholerae) and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in an inland locality, which is known as endemic for cholera, and compared them with those of a cholera-free coastal location. We found the consistent presence of the pandemic-generating lineage of V. cholerae in Dhaka, where cholera is endemic, and an exclusive presence of a lineage phylogenetically distinct from other V. cholerae lineages. Our study suggests that this lineage represents a novel species that has pathogenic potential and a human link to its environmental abundance. The possible association with human populations and coexistence and interaction with toxigenic V. cholerae in the natural environment make this potential human pathogen an important subject for future studies.

Simultaneous Quantification of Vibrio metoecus and Vibrio cholerae with Its O1 Serogroup and Toxigenic Subpopulations in Environmental Reservoirs.

Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.