A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used ...

Hiroshima University

To study the size-resolved characteristics of airborne bacterial community composition, diversity, and abundance, outdoor aerosol samples were analysed by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing with Illumina MiSeq. The samples were collected using size-resolved samplers between August and October 2016, at a suburban site in Toyama City and an urban site in Yokohama City, Japan. The bacterial communities were found to be dominated by Actinobacteria, Firmicutes, and Proteobacteria. At the genus level, we found a high abundance of human skin-associated bacteria, such as Propionibacterium, Staphylococcus, and Corynebacterium, in the urban site. Whereas, a high abundance of bacteria associated with soil and plants, such as Methylobacterium and Sphingomonas, was observed in the suburban site. Furthermore, our data revealed a shift in the bacterial community structure, diversity, and abundance of total bacteria at a threshold of 1.1-µm diameter. Interestingly, we observed that Legionella spp., the causal agents of legionellosis in humans, were mainly detected in > 2.1 µm coarse particles. Our data indicate that local environmental factors including built environments could influence the outdoor airborne bacterial community at each site. These results provide a basis for understanding the size-resolved properties of bacterial community composition, diversity, and abundance in outdoor aerosol samples and their potential influence on human health.

Size resolved characteristics of urban and suburban bacterial bioaerosols in Japan as assessed by 16S rRNA amplicon sequencing.

MinION (Oxford Nanopore Technologies), a portable nanopore sequencer, was introduced in 2014 as a new DNA sequencing technology. MinION is now widely used because of its low initial start-up costs relative to existing DNA sequencers, good portability, easy-handling, real-time analysis and long-read output. However, differences in the experimental conditions used for 16S rRNA-based PCR can bias bacterial community assessments in samples. Therefore, basic knowledge about reliable experimental conditions is needed to ensure the appropriate use of this technology. Our study concerns the reliability of techniques for obtaining accurate and quantitative full-length 16S rRNA amplicon sequencing data for bacterial community structure assessment using MinION. We compared five PCR conditions using three independent mock microbial community standard DNAs and established appropriate, standardized, better PCR conditions among the trials. We then sequenced two mock communities and six environmental samples using Illumina MiSeq for comparison. Modifying the PCR conditions improved the sequencing quality; the optimized conditions were 35 cycles of 95 °C for 1 min, 60 °C for 1 min and 68 °C for 3 min. Our results provide important information for researchers to determine bacterial community using MinION accurately.

Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer.

Microbial entities (such bacteria, fungi, archaea and viruses) within outdoor aerosols have been scarcely studied compared with indoor aerosols and nonbiological components, and only during the last few decades have their studies increased. Bacteria represent an important part of the microbial abundance and diversity in a wide variety of rural and urban outdoor bioaerosols. Currently, airborne bacterial communities are mainly sampled in two aerosol size fractions (2.5 and 10 µm) and characterized by culture-dependent (plate-counting) and culture-independent (DNA sequencing) approaches. Studies have revealed a large diversity of bacteria in bioaerosols, highlighting Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as ubiquitous phyla. Seasonal variations in and dispersion of bacterial communities have also been observed between geographical locations as has their correlation with specific atmospheric factors. Several investigations have also suggested the relevance of airborne bacteria in the public health and agriculture sectors as well as remediation and atmospheric processes. However, although factors influencing airborne bacterial communities and standardized procedures for their assessment have recently been proposed, the use of bacterial taxa as microbial indicators of specific bioaerosol sources and seasonality have not been broadly explored. Thus, in this review, we summarize and discuss recent advances in the study of airborne bacterial communities in outdoor environments and the possible factors influencing their abundance, diversity, and seasonal variation. Furthermore, airborne bacterial activity and bioprospecting in different fields (e.g., the textile industry, the food industry, medicine, and bioremediation) are discussed. We expect that this review will reveal the relevance and influencing factors of airborne bacteria in outdoor environments as well as stimulate new investigations on the atmospheric microbiome, particularly in areas where air quality is a public concern.

Airborne bacterial communities of outdoor environments and their associated influencing factors.

Harmful algae blooms (HABs) cause acute effects on marine ecosystems due to their production of endogenous toxins or their enormous biomass, leading to significant impacts on local economies and public health. Although HAB monitoring has been intensively performed at spatiotemporal scales in coastal areas of the world over the last decades, procedures have not yet been standardized. HAB monitoring procedures are complicated and consist of many methodologies, including physical, chemical, and biological water sample measurements. Each monitoring program currently uses different combinations of methodologies depending on site specific purposes, and many prior programs refer to the procedures in quotations. HAB monitoring programs in Chile have adopted the traditional microscopic and toxin analyses but not molecular biology and bacterial assemblage approaches. Here we select and optimize the HAB monitoring methodologies suitable for Chilean geography, emphasizing on metabarcoding analyses accompanied by the classical tools with considerations including cost, materials and instrument availability, and easiness and efficiency of performance. We present results from a pilot study using the standardized stepwise protocols, demonstrating feasibility and plausibility for sampling and analysis for the HAB monitoring. Such specific instructions in the standardized protocol are critical obtaining quality data under various research environments involving multiple stations, different analysts, various time-points, and long HAB monitoring duration.

Protocols for Monitoring Harmful Algal Blooms for Sustainable Aquaculture and Coastal Fisheries in Chile.

Phytoplankton blooms, including harmful algal blooms (HABs), have serious impacts on ecosystems, public health, and productivity activities. Rapid detection and monitoring of marine microalgae are important in predicting and managing HABs. We developed a toolkit, the Suitcase Lab, to detect harmful algae species in the field. We demonstrated the Suitcase Lab's capabilities for sampling, filtration, DNA extraction, and loop-mediated isothermal amplification (LAMP) detection in cultured Alexandrium catenella cells as well as Chilean coastal waters from four sites: Repollal, Isla García, Puerto Montt, and Metri. A LAMP assay using the Suitcase Lab in the field confirmed microscopic observations of A. catenella in samples from Repollal and Isla García. The Suitcase Lab allowed the rapid detection of A. catenella, within 2 h from the time of sampling, even at a single cell per milliliter concentrations, demonstrating its usefulness for quick and qualitative on-site diagnosis of target toxic algae species. This method is applicable not only to detecting harmful algae but also to other field studies that seek a rapid molecular diagnostic test.

Suitcase Lab: new, portable, and deployable equipment for rapid detection of specific harmful algae in Chilean coastal waters.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

So Fujiyoshi

Office of Research and Academia-Government-Community Collaboration

Hiroshima University

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

So Fujiyoshi

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Hiroshima University

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

Office of Research and Academia-Government-Community Collaboration