16S Next-generation sequencing and quantitative PCR reveal the distribution of potential pathogens in the Liaohe Estuary

Abstract

The existence of potentially pathogenic bacteria seriously threatens aquatic animals and human health. Estuaries are closely related to human activities, and the detection of pathogens is important for aquaculture and public health. However, monitoring only indicator microorganisms and pathogens is not enough to accurately and comprehensively estimate water pollution. Here, the diversity of potentially pathogenic bacteria in water samples from the Liaohe estuary was profiled using 16S next-generation sequencing (16S NGS) and quantitative polymerase chain reaction (qPCR) analysis. The results showed that the dominant genera of environmental pathogens were Pseudomonas, Vibrio, Mycobacterium, Acinetobacter, Exiguobacterium, Sphingomonas, and Legionella, and the abundance of enteric pathogens was significantly less than the environmental pathogens, mainly, Citrobacter, Enterococcus, Escherichia-Shigella, Enterobacter, Bacteroides. The qPCR results showed that the 16S rRNA genes of Vibrio were the most abundant, with concentrations between 7.06 and 9.48 lg copies/L, followed by oaa gene, fliC gene, trh gene, and uidA gene, and the temperature and salinity were the main factors affecting its abundance. Variance partitioning analysis (VPA) analysis of spatial factors on the potential pathogen’s distribution (19.6% vs 5.3%) was greater than environmental factors. In addition, the co-occurrence analysis of potential pathogens in the estuary revealed significant co-occurrence among the opportunistic pathogens Testosteronemonas, Brevimonas vesicularis, and Pseudomonas putida. Our findings provide an essential reference for monitoring and occurrence of potentially pathogenic bacteria in estuaries.

Tools to Enumerate and Predict Distribution Patterns of Environmental Vibrio vulnificus and Vibrio parahaemolyticus

“…In addition to all the environmental factors described above, introduction of microplastics and heavy metals into waterbodies near aquaculture facilities may also increase abundances. For example, Vp enumerated with trh qPCR was positively correlated with heavy metals in water samples, but the impact of metals was also seen for general bacteria, E. coli, Pseudomonas aeruginosa, among other groups [134]. Additionally, growth and biofilm formation by both pathogenic and non-pathogenic types of Vv are positively correlated with increasing iron concentrations [173].…”

“…There is little "correlation" of Vibrio with SST when short-term, within-season, evaluations are conducted in both culture-dependent [128] and culture-independent [134,142] types of studies. This is especially evident from studies where the entire sampling period occurs in the warm summer months [134] or from single-year evaluations where temperature is a strong factor [143].…”

“…There is little "correlation" of Vibrio with SST when short-term, within-season, evaluations are conducted in both culture-dependent [128] and culture-independent [134,142] types of studies. This is especially evident from studies where the entire sampling period occurs in the warm summer months [134] or from single-year evaluations where temperature is a strong factor [143]. Long-term, there are positive relationships between increased temperature with Vibrio in the zooplankton and phytoplankton seawater fraction [122], in the water column [112], and with Vp associated with shellfish [106].…”

“…Sensitivity of qPCR-only studies is, like with methods containing a culture step, very good. For example, Vp detection is as low as 1 to 10 copies per mL of seawater with trh primers [134]; with these and other primers for Vp and for other Vibrio species, a similar range is detectable with the very sensitive qPCR method [137]. Detection of Vv with direct qPCR is also very sensitive; cell equivalents in DNA enumerated with vvhA qPCR is as low as 15 CFU per mg of homogenized fish tissue [54].…”

Seasonal Dynamics of Marine Bacterial Communities in Aquaculture Farms: The case of the Northern Ionian Coastal Ecosystem (Mediterranean Sea)

“…The assessment of bacterial diversity and the respective interactions in aquaculture systems can be achieved by next-generation sequencing (NGS) technologies, particularly with the use of 16S rRNA gene amplicon sequencing [21,22]. This technology has been effectively used to study the temporal dynamics of marine microbial communities in aquaculture farms [23], identify the presence of potential pathogens [24,25], and assess the impact of environmental stressors, such as climate change, mainly in ecosystems like the Mediterranean Sea where large variations in future temperature levels are expected [26].…”