Vibrio Ecology in the Neuse River Estuary, North Carolina, Characterized by Next-Generation Amplicon Sequencing of the Gene Encoding Heat Shock Protein 60 ( hsp60 )

Abstract

Of marine eubacteria, the genus is intriguing because member species are relevant to both marine ecology and human health. Many studies have touted the relationships of to environmental factors, especially temperature and salinity, to predict total abundance but lacked the taxonomic resolution to identify the relationships among species and the key drivers of dynamics. To improve next-generation sequencing (NGS) surveys of, we have conducted both 16S small subunit rRNA and heat shock protein 60 () amplicon sequencing of water samples collected at two well-studied locations in the Neuse River Estuary, NC. Samples were collected between May and December 2016 with enhanced sampling efforts in response to two named storms. Using sequences, 21 species were identified, including the potential human pathogens ,, and Changes in the community mirrored seasonal and storm-related changes in the water column, especially in response to an influx of nutrient-rich freshwater to the estuary after Hurricane Matthew, which initiated dramatic changes in the overall community. Individual species dynamics were wide ranging, indicating that individual taxa have unique ecologies and that total abundance predictors are insufficient for risk assessments of potentially pathogenic species. Positive relationships between, dinoflagellates, and were identified, as were intraspecies associations, which further illuminated the interactions of cooccurring taxa along environmental gradients. The objectives of this research were to utilize a novel approach to improve sequence-based surveys of communities and to demonstrate the usefulness of this approach by presenting an analysis of dynamics in the context of environmental conditions, with a particular focus on species that cause disease in humans and on storm effects. The methods presented here enabled the analysis of dynamics with excellent taxonomic resolution and could be incorporated into future ecological studies and risk prediction strategies for potentially pathogenic species. Next-generation sequencing of and other innovative sequence-based approaches are valuable tools and show great promise for studying ecology and associated public health risks.

Spatial Heterogeneity of Vibrio spp. in Sediments of Chinese Marginal Seas

“…However, a higher proportion of community variation was explained by the joint effect of the two types of factors. Thus, our study expanded upon previous studies, where only the influence of environmental factors was investigated (1,4,7,18), by showing an interaction of spatial and environmental variables in determining the distribution of Vibrio communities. These interactions include the latitude-dependent change in temperature.…”

“…It was reported that next-generation sequencing (NGS) of protein-coding marker genes, including those encoding heat shock protein 60 (hsp60) (1), ferric uptake regulator (fur) (70), and uridylate kinase (pyrH) (18), may replace the 16S rRNA gene as excellent markers for differentiating closely related Vibrio spp. (1). However, it is difficult to amplify environmental Vibrio spp.…”

“…act as potential pathogens (5,6). Many well-known Vibrio spp., such as Vibrio cholerae, V. parahaemolyticus, V. vulnificus, V. anguillarum, and V. harveyi, cause disease in humans and/or marine animals (1,7).…”

“…Most earlier studies were carried out by culture-based techniques (18,22,23), which are problematic due to the high frequency of Vibrio bacteria entering a viable but nonculturable (VBNC) state (24,25). More recently, the distribution pattern of Vibrio populations has been investigated by quantitative PCR (qPCR) and high-throughput sequencing on the basis of Vibrio-specific 16S rRNA gene primers (1,7,26). Typically, the abundance of Vibrio spp.…”

A New High Throughput Sequencing Assay for Characterizing the Diversity of Natural Vibrio Communities and Its Application to a Pacific Oyster Mortality Event

“…Due to the inherent inadequacies of the 16S rRNA gene for correctly identifying Vibrio species, another gene target encoding the heat shock protein 60 (hsp60, also known as groEL or cpn60), a type I chaperonin protein that assists in protein folding (Hemmingsen et al, 1988;Farr et al, 2000;Brinker et al, 2001), has been proposed as a good candidate for Vibrio phylogenetic studies (Kwok et al, 2002). Previous studies utilizing hsp60 have primarily relied on Vibrio isolates for species identification (Preheim et al, 2011;Szabo et al, 2012;Silvester et al, 2017), but a recent study applied universal hsp60 primers to characterize Vibrio community diversity using high throughput amplicon sequencing (Jesser and Noble, 2018). While this approach delivered improved Vibrio species identification relative to 16S rRNA, it used universal, rather than Vibrio-centric, hsp60 primers and required bioinformatic filtering for Vibrio assigned species, as is often required for 16S rRNA sequencing.…”