Microbial Communities Associated with Farmed Genypterus chilensis: Detection in Water Prior to Bacterial Outbreaks Using Culturing and High-Throughput Sequencing.

The red conger eel (Genypterus chilensis, Guichenot) is a native species included in the Chilean Aquaculture Diversification Program due to high commercial demand. In the context of intensified farming, prior reports link two disease outbreaks with emerging pathogens in the Vibrio and Tenacibaculum genera. However, the roles remain unclear for the bacterial community and each specific bacterium is associated with the rearing environment for healthy specimens. The success of red conger eel farming therefore warrants research into the bacterial composition of aquaculture conditions and the antimicrobial susceptibilities thereof. This study used culturing methods and high-throughput sequencing to describe the bacterial community associated with water in which G. chilensis was farmed. With culturing methods, the predominant genera were Vibrio (21.6%), Pseudolteromonas (15.7%), Aliivibrio (13.7%), and Shewanella (7.8%). Only a few bacterial isolates showed amylase, gelatinase, or lipase activity, and almost all showed inhibition zones to commonly-used antibiotics in aquaculture. By contrast, high-throughput sequencing established Paraperlucidibaca, Colwellia, Polaribacter, Saprospiraceae, and Tenacibaculum as the predominant genera, with Vibrio ranking twenty-seventh in abundance. High-throughput sequencing also established a link between previous outbreaks with increased relative abundances of Vibrio and Tenacibaculum. Therefore, monitoring the presence and abundance of these potential pathogens could be useful in providing prophylactic measures to prevent future outbreaks.

Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups.

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

A single genus in the gut microbiome reflects host preference and specificity.

Delineating differences in gut microbiomes of human and animal hosts contributes towards understanding human health and enables new strategies for detecting reservoirs of waterborne human pathogens. We focused upon Blautia, a single microbial genus that is important for nutrient assimilation as preliminary work suggested host-related patterns within members of this genus. In our dataset of 57 M sequence reads of the V6 region of the 16S ribosomal RNA gene in samples collected from seven host species, we identified 200 high-resolution taxonomic units within Blautia using oligotyping. Our analysis revealed 13 host-specific oligotypes that occurred exclusively in fecal samples of humans (three oligotypes), swine (six oligotypes), cows (one oligotype), deer (one oligotype), or chickens (two oligotypes). We identified an additional 171 oligotypes that exhibited differential abundance patterns among all the host species. Blautia oligotypes in the human population obtained from sewage and fecal samples displayed remarkable continuity. Oligotypes from only 10 Brazilian human fecal samples collected from individuals in a rural village encompassed 97% of all Blautia oligotypes found in a Brazilian sewage sample from a city of three million people. Further, 75% of the oligotypes in Brazilian human fecal samples matched those in US sewage samples, implying that a universal set of Blautia strains may be shared among culturally and geographically distinct human populations. Such strains can serve as universal markers to assess human fecal contamination in environmental samples. Our results indicate that host-specificity and host-preference patterns of organisms within this genus are driven by host physiology more than dietary habits.

The microbiome of urban waters.

More than 50% of the world's population lives in urban centers. As collection basins for landscape activity, urban waters are an interface between human activity and the natural environment. The microbiome of urban waters could provide insight into the impacts of pollution, the presence of human health risks, or the potential for long-term consequences for these ecosystems and the people who depend upon them. An integral part of the urban water cycle is sewer infrastructure. Thousands of miles of pipes line cities as part of wastewater and stormwater systems. As stormwater and sewage are released into natural waterways, traces of human and animal microbiomes reflect the sources and magnitude of fecal pollution and indicate the presence of pollutants, such as nutrients, pathogens, and chemicals. Non-fecal organisms are also released as part of these systems. Runoff from impervious surfaces delivers microbes from soils, plants and the built environment to stormwater systems. Further, urban sewer infrastructure contains its own unique microbial community seemingly adapted to this relatively new artificial habitat. High microbial densities are conveyed via pipes to waterways, and these organisms can be found as an urban microbial signature imprinted on the natural community of rivers and urban coastal waters. The potential consequences of mass releases of non-indigenous microorganisms into natural waters include creation of reservoirs for emerging human pathogens, altered nutrient flows into aquatic food webs, and increased genetic exchange between two distinct gene pools. This review highlights the recent characterization of the microbiome of urban sewer and stormwater infrastructure and its connection to and potential impact upon freshwater systems.

Blautia and Prevotella sequences distinguish human and animal fecal pollution in Brazil surface waters.

Untreated sewage discharges and limited agricultural manure management practices contribute to fecal pollution in rural Brazilian waterways. Most microbial source tracking studies have focused on Bacteroidales, and few have tested host-specific indicators in underdeveloped regions. Sequencing of sewage and human and animal feces with Illumina HiSeq revealed Prevotellaceae as the most abundant family in humans, with Lachnospiraceae and Ruminococcaceae also comprising a large proportion of the microbiome. These same families were also dominant in animals. Bacteroides, the genus containing the most commonly utilized human-specific marker in the United States was present in very low abundance. We used oligotyping to identify Prevotella and Blautia sequences that can distinguish human fecal contamination. Thirty-five of 61 Blautia oligotypes and 13 of 108 Prevotella oligotypes in humans were host-specific or highly abundant (i.e. host-preferred) compared to pig, dog, horse and cow sources. Certain human Prevotella and Blautia oligotypes increased more than an order of magnitude along a polluted river transect in rural Brazil, but traditional fecal indicator levels followed a steady or even decreasing trend. While both Prevotella and Blautia oligotypes distinguished human and animal fecal pollution in Brazil surface waters, Blautia appears to contain more discriminatory and globally applicable markers for tracking sources of fecal pollution.

Sources and distribution of surface water fecal contamination and prevalence of schistosomiasis in a Brazilian village.

BACKGROUND: The relationship between poor sanitation and the parasitic infection schistosomiasis is well-known, but still rarely investigated directly and quantitatively. In a Brazilian village we correlated the spatial concentration of human fecal contamination of its main river and the prevalence of schistosomiasis. METHODS: We validated three bacterial markers of contamination in this population by high throughput sequencing of the 16S rRNA gene and qPCR of feces from local residents. The qPCR of genetic markers from the 16S rRNA gene of Bacteroides-Prevotella group, Bacteroides HF8 cluster, and Lachnospiraceae Lachno2 cluster as well as sequencing was performed on georeferenced samples of river water. Ninety-six percent of residents were examined for schistosomiasis. FINDINGS: Sequence of 16S rRNA DNA from stool samples validated the relative human specificity of the HF8 and Lachno 2 fecal indicators compared to animals. The concentration of fecal contamination increased markedly along the river as it passed an increasing proportion of the population on its way downstream as did the sequence reads from bacterial families associated with human feces. Lachnospiraceae provided the most robust signal of human fecal contamination. The prevalence of schistosomiasis likewise increased downstream. Using a linear regression model, a significant correlation was demonstrated between the prevalence of S. mansoni infection and local concentration of human fecal contamination based on the Lachnospiraceae Lachno2 cluster (r2 0.53) as compared to the correlation with the general fecal marker E. coli (r2 0.28). INTERPRETATION: Fecal contamination in rivers has a downstream cumulative effect. The transmission of schistosomiasis correlates with very local factors probably resulting from the distribution of human fecal contamination, the limited movement of snails, and the frequency of water contact near the home. In endemic regions, the combined use of human associated bacterial markers and GIS analysis can quantitatively identify areas with risk for schistosomiasis as well as assess the efficacy of sanitation and environmental interventions for prevention.

The polycyclic aromatic hydrocarbon degradation potential of Gulf of Mexico native coastal microbial communities after the Deepwater Horizon oil spill.

The Deepwater Horizon (DWH) blowout resulted in oil transport, including polycyclic aromatic hydrocarbons (PAHs) to the Gulf of Mexico shoreline. The microbial communities of these shorelines are thought to be responsible for the intrinsic degradation of PAHs. To investigate the Gulf Coast beach microbial community response to hydrocarbon exposure, we examined the functional gene diversity, bacterial community composition, and PAH degradation capacity of a heavily oiled and non-oiled beach following the oil exposure. With a non-expression functional gene microarray targeting 539 gene families, we detected 28,748 coding sequences. Of these sequences, 10% were uniquely associated with the severely oil-contaminated beach and 6.0% with the non-oiled beach. There was little variation in the functional genes detected between the two beaches; however the relative abundance of functional genes involved in oil degradation pathways, including polycyclic aromatic hydrocarbons (PAHs), were greater in the oiled beach. The microbial PAH degradation potentials of both beaches, were tested in mesocosms. Mesocosms were constructed in glass columns using sands with native microbial communities, circulated with artificial sea water and challenged with a mixture of PAHs. The low-molecular weight PAHs, fluorene and naphthalene, showed rapid depletion in all mesocosms while the high-molecular weight benzo[α]pyrene was not degraded by either microbial community. Both the heavily oiled and the non-impacted coastal communities showed little variation in their biodegradation ability for low molecular weight PAHs. Massively-parallel sequencing of 16S rRNA genes from mesocosm DNA showed that known PAH degraders and genera frequently associated with oil hydrocarbon degradation represented a major portion of the bacterial community. The observed similar response by microbial communities from beaches with a different recent history of oil exposure suggests that Gulf Coast beach communities are primed for PAH degradation.