Chapter 4. Monitoring Bacteria with their Antibiotic-Resistant Genes (ARGs) and Interaction between
4.2. Materials and methods
4.2.1. Sampling
In total, 28 seawater samples from a depth of approximately 7 m were obtained during the R/V Aaron icebreaker (Korea Polar Research Institute, Korea) voyage from 31 October to 4 December 2019 (35 days) in the western Pacific Ocean and the Southern Ocean. The total distance of the cruise was 14,942 km, and the sampling sites are shown in Fig. 4.1. Exclusive economic zones (EEZs) were excluded during sampling, and distances between each sampling stations and adjacent land are shown in Table 4.1.
Sampling in the western Pacific Ocean was conducted at 13 sites (from ST01 to ST13, Fig. 4.1) over a distance of 10,751 km, covering the West Sea of Korea (WS), the Philippine Sea (PS), Western Equatorial Pacific Ocean (WEPO), and Tasman Sea (TS) in the western Pacific Ocean. Sampling at ST01–03 was conducted in the WS immediately after departure from Incheon Port (Korea) on 31 October 2019. ST03 was situated closer to South Korea (Jeju Island) and only 208 km away from Japan;
therefore, we hypothesized that this station would provide samples contaminated by pollutants from both Korea and Japan. Samples were collected at three sites near the PS (ST04–06), situated 1,625 ± 57 km away from the Philippines islands. Three other sites (ST07–09) near the WEPO were selected for sample collection, and these sites were possibly influenced by Papua New Guinea. The last four samples of Cruise 1 were collected between the TS (ST10–13). The TS is located an average of 1,126 km from Australia and New Zealand. All sites in the western Pacific Ocean were situated approximately 51–
1,662 km (average 850 km) away from the nearest land (Table 4.1).
In the Southern Ocean, 15 samples (ST14–ST28, Fig. 4.1) were collected during a 4,191 km long cruise, including the New Zealand sector of the Southern Ocean (NZSSO) and Ross Sea (RS). Sampling on the R/V Araon, which departed from Littleton Port, New Zealand, on 25 November 2019, was performed outside the New Zealand’s EEZ (ST14–18; 1,836 ± 676 km away from New Zealand). The Jangbogo Station is located in the Terra Nova Bay, west of southeast Antarctica, and the stations of Ge-
Fig. 4.1. (a) Geographic distribution of surface seawater temperature and total ARG abundance at sampling stations. ST01–03 (West Sea of Korea; WS, purple), ST04–06 (Philippine Sea; PS, blue), ST07–09 (Western Equatorial Pacific Ocean; WEPO, red), ST10–13 (Tasman Sea; TS, yellow), ST14–24, ST39–44 (New Zealand sector of the Southern Ocean; NZSSO; green), and ST25–28 (Ross Sea; RS, navy). (b) The images and (c) the schematic diagram of experimental sea spray aerosol generation chamber system on board R/V Araon.
Table 4.1. Water quality parameters and distance from adjacent countries at each sampling station.
Region Station Sampling date
(UTC) Latitude Longitude
Water temperature
(°C)
Salinity
(PSU) Adjacent country Distance from the country (km)
Western Pacific Ocean
West Sea of Korea
(WS)
ST01 2019.10.31.
(13:16) 36°13N 125°50E 18.67 31.88 South Korea 60.72
China 304.14
ST02 2019.11.01.
(00:02) 33°54N 125°48E 19.60 32.76
South Korea 77.84
Japan 281.66
China 433.56
ST03 2019.11.01.
(06:04) 32°46N 126°23E 24.75 33.98
South Korea
(Jeju Island) 51.39
Japan 207.64
China 431.82
Philippine Sea (PS)
ST04 2019.11.05.
(00:00) 17°14N 138°03E 29.59 34.23 Philippines 1654.79
ST05 2019.11.05.
(12:01) 15°06N 139°35E 29.98 34.40 Philippines 1559.26
ST06 2019.11.05.
(23:30) 13°06N 141°01E 30.59 33.99 Philippines 1661.66
Western Equatorial
Pacific Ocean (WEPO)
ST07 2019.11.08.
(03:00) 04°16N 147°29E 30.27 33.97 Papua New Guinea 960.68
ST08 2019.11.08.
(07:59) 03°25N 148°08E 30.06 33.92 Papua New Guinea 916.97
ST09 2019.11.08.
(13:00) 02°32N 148°49E 30.54 34.07 Papua New Guinea 868.54
Tasman Sea (TS)
ST10 2019.11.14.
(20:21) 24°25S 163°01E 24.16 35.64 Australia 1007.85
New Zealand 1453.44
ST11 2019.11.15.
(19:09) 28°46S 163°51E 22.60 35.70 Australia 998.62
New Zealand 1044.41
ST12 2019.11.16.
(19:00) 33°04S 165°49E 20.80 35.68 New Zealand 650.92
ST13 2019.11.17.
(18:49) 37°05S 168°53E 18.32 35.42 New Zealand 428.24
(continued)
Southern Ocean
New Zealand sector of
the Southern
Ocean (NZSSO)
ST14 2019.11.26.
(19:51) 53°04S 178°18E 9.33 33.84 New Zealand 960.53
ST15 2019.11.27.
(03:34) 54°31S 179°13E 9.62 33.87 New Zealand 1118.02
ST16 2019.11.27.
(11:00) 55°50S 179°27E 9.02 33.85 New Zealand 1240.62
ST17 2019.11.27.
(19:00) 57°09S 177°36E 7.80 33.77 New Zealand 1302.25
ST18 2019.11.28.
(07:03) 59°04S 174°59E 6.90 33.60 New Zealand 1437.13
ST19 2019.11.28.
(19:03) 61°30S 174°10E 4.59 33.54 New Zealand 1681.35
ST20 2019.11.29.
(07:04) 63°47S 173°17W 1.74 33.32 New Zealand 2190.23
ST21 2019.11.29.
(14:09) 65°09S 172°44W 0.43 33.41 New Zealand 2326.20
ST22 2019.11.29.
(18:29) 66°00S 172°19W -0.18 33.50 New Zealand 2412.24
ST23 2019.11.30.
(07:17) 68°11S 171°28W 2.27 33.65 New Zealand 2632.76
ST24 2019.12.01.
(03:04) 71°05S 171°54W 1.11 34.09 New Zealand 2898.66
Ross Sea (RS)
ST25 2019.12.02.
(16:19) 74°45S 177°41E 0.70 34.18 Antarctica 299.52
ST26 2019.12.03.
(00:33) 75°15S 171°48E 1.25 34.20 Antarctica 196.94
ST27 2019.12.03.
(07:29) 74°55S 166°41E 0.86 33.95 Antarctica 47.97
ST29 2019.12.04.
(21:19) 74°37S 164°14E 0.45 34.55 Antarctica 0.42
ST31 2019.12.06
(20:02) 74°37S 164°14E - 34.43 Antarctica 0.42
ST35 2019.12.08
(21:00) 74°42S 164°34E 1.87 34.59 Antarctica 11.14
ST36 2019.12.09
(0:40) 74°57S 167°16E 1.68 34.33 Antarctica 93.86
ST37 2019.12.09
(8:01) 74°59S 172°55E 1.46 33.96 Antarctica 254.70
(continued)
Southern Ocean
New Zealand sector of
the Southern
Ocean (NZSSO)
ST39 2019.12.10
(19:39) 70°42S 171°01W - 33.92 New Zealand 2882.94
ST41 2019.12.11
(19:43) 68°36S 172°40W 1.48 34.01 New Zealand 2382.87
ST42 2019.12.12
(0:01) 67°02S 178°16W 0.61 33.76 New Zealand 2307.13
ST43 2019.12.15
(0:00) 66°33S 179°49W 0.75 33.80 New Zealand 1995.85
ST44 2019.12.18
(23:08) 64°13S 175°25E - 34.07 New Zealand 1694.72
rmany (Gondwana Camp) and Italy (Mario Zucchelli Station) operate within a 10 km radius of the Jangbogo Station. Sea ice occurred after ST19, and ST25 was located in polynya. In general, the sampling sites in the RS were between 0.42 km (ST28; Jangbogo Station) and 299 km (ST25) away from Antarctica (Table 4.1).
Bulk seawater samples from each location were collected at an approximate depth of 7 m in flexible plastic containers using an in-vessel pump. The containers were washed sequentially with 70% ethanol, ultrapure water, and seawater immediately before each sample was collected. After sampling, between 150 mL and 4 L of seawater was filtered through a membrane filter (0.45 μm, nylon, Whatman) directly through a vacuum filter system for pretreatment to quantify the ARGs. For bacterial diversity analysis, total bacterial cells were trapped in 3.0 μm and 0.2 μm membrane filters under a vacuum, for particle- attached bacteria and free-living bacteria, respectively. The filters were sterilized with an autoclave before use and kept on an in-vessel clean bench. After filtration, each filter was stored in a 2-mL e-tube (Axygen, USA) at −80 °C for DNA extraction, and the blank filters were stored using the same storage methods without filtration. All samples were transferred to a laboratory with dry ice via a refrigeration van after unloading in Korea. At each sampling stations, each 2 filters were collected for ARGs, particle- attached and free-living bacteria and each 1 filter for blank. The environmental parameters at each station, including water temperature and salinity were measured using a probe to determine the conductivity–temperature–depth profile of the vessel (Table 4.1). The distance between each station to the nearest land was measured based on latitude and longitude in Google Earth pro (ver. 7.3.3.7786).