A Study on the Marine Prokaryotic Diversity and Antibiotic Resistome using High-Throughput Sequencing and Functional Metagenomic Analysis

Abstract

Prokaryotes (Bacteria and Archaea) play a significant role in global biogeochemical flux of biologically important elements in marine ecosystems. It has been firmly established that prokaryotes are major primary producers and heterotrophic consumers. Despite prokaryotes have significant roles in the biogeochemical flux in marine environmets, it has been estimated that more than 99% of the bacteria are non-culturable, leading to limited information of prokaryotic diversity because of an inability to mimic proper environmental niches. The relation between prokaryotic communities and their roles in the biogeochemical cycle is a topic of central research in environmental microbiology. Knowledge of the prokaryotic community compositions has rapidly increased due to development of molecular techniques based on amplification of the 16S rRNA gene and sequencing. This thesis includes studies on i) seasonal and spatial distribution of diversity of marine prokaryotes in the East Sea using pyrosequencing, ii) marine bacteria can be dispersed in the atmospheric environments, and iii) diversity of antibiotic resistance genes in the water column of the East Sea using the functional metagenomics approach.

i) To understand in-depth prokaryotic diversity using high-throughput sequencing technique, we applied a pyrosequencing approach to study spatial and seasonal distribution of prokaryotes in the East Sea during 2011-2014. This study demonstrates that bacterial and archaeal communities varied along horizontal scale from coastal to offshore as well as vertical scale along depth, and furthermore their distribution patterns were different between seasons. Bacteroidetes, Alpha- and Gamma-proteobacteria dominated from epipelagic to mesopelagic zones in the East Sea. However, the discrepancy must be resulted from the low coverage of used primer for a major Alpha-proteobacteria group (SAR11). Thaumarchaeota and Euryarchaeota were dominant archaea in the epipelagic zone (0-100 m). Euryarchaeota decreased gradually with increasing depth. We found that dominant archaeal groups such as Nitrosopumilaceae and Halobacteria were more dynamics of distribution patterns in the epipelagic zone than in the mesopelagic zone.

ii) Rainwater is an important natural resource to study airborne marine bacteria. Rainwater samples were collected during three heavy rain events at a suburban site in Seoul. Bacterial community compositions (BCCs) of rainwater samples, analyzed by using 16S rRNA gene-based pyrosequencing, differed considerably among the three rain events. Presumable marine bacterial OTUs which formed a robust clade with marine bacteria Lacinutrix spp. were at high concentrations in rainwater in April, likely reflecting origin from saline environments. Most of the Flavobacteria sequences were unusually high in April rainwater, which presumed to be of marine origin. Thus, these results suggest that some marine bacteria can be disseminated as aerosol particles and precipitated on land via rain.

iii) The antibiotic resistance (AR) issue has critically increased. With the increasing use of antibiotics, resistance to antibiotics is developed by bacteria having AR mechanisms to defend themselves and survive from the antibiotic-polluted environments. Functional metagenomic approach is more useful to discover novel AR genes and resistance mechanisms rather than culture-dependent approaches, PCR and sequencing methods. We found a variety of AR genes conferring resistance to ampicillin, polymyxin B, rifampicin, fosfomycin and gentamicin in the East Sea. Among antibiotics, the majority (53.4%) of the cloned resistant genes was resistant to ampicillin, 18.8% to polymyxin B, 16.1% to rifampicin, 7.2% to fosfomycin and 4.5% to gentamicin. The source of host organisms inferred from the annotated AR genes was mainly marine bacterial taxa.

This thesis suggested that bacteria might play a more significant role in marine environments. Bacterial community compositions in the East Sea varied across space and seasons. Furthermore, some marine bacteria can disperse in the atmospheric environments. Marine environments may be potentially global reservoir of AR genes.