S1 Journal: Environment International Supplementary materials of the article: Large-scale biogeographical patterns of bacterial antibiotic resistome in the waterbodies of China Lemian Liu a,b,# , Jian-Qiang Su a,# , Yunyan Guo a,c,# , David M. Wilkinson d , Zhengwen Liu e , Yong-Guan Zhu a , Jun Yang a, * a Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China b College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, People’s Republic of China c University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China d School of Life Sciences, University of Lincoln, Lincoln LN6 7TS, UK. e State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, People’s Republic of China # These authors contributed equally to this work. *Corresponding author: Jun Yang; E-mail: [email protected]This supplementary information contains: 36 Pages 6 Figures 11 Tables 47 References
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S1
Journal: Environment International
Supplementary materials of the article:
Large-scale biogeographical patterns of bacterial
antibiotic resistome in the waterbodies of China
Lemian Liu a,b,#, Jian-Qiang Su a,#, Yunyan Guo a,c,#, David M. Wilkinson d,
Zhengwen Liu e, Yong-Guan Zhu a, Jun Yang a,*
a Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese
Academy of Sciences, Xiamen 361021, People’s Republic of China b College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, People’s
Republic of China c University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China d School of Life Sciences, University of Lincoln, Lincoln LN6 7TS, UK. e State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and
Limnology, Chinese Academy of Sciences, Nanjing 210008, People’s Republic of China
gene copy number, right) of ARGs in five geographical regions across China (data are expressed
as mean ± s.e.). FJ (included 5 reservoirs) - Fujian province, southeast China; CJ (9 lakes) - the
lower and middle reaches of Changjiang River, China; ECC (6 lakes) - east central China, IM (13
lakes) - Inner Mongolia, north China; NEC (9 lakes) - northeast China. Others - the ARGs
conferring resistance to other antibiotic classes, including bacitracin, fosfomycin, imipenem,
lantibiotic, nitroimidazole, pyrazinamide, triclosan, trimethoprim and unknown.
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Fig. S4 Richness of ARGs in five geographical regions across China (data are expressed as mean
± s.e.). FJ (included 5 reservoirs) - Fujian province, southeast China; CJ (9 lakes) - the lower and
middle reaches of Changjiang River, China; ECC (6 lakes) - east central China, IM (13 lakes) -
Inner Mongolia, north China; NEC (9 lakes) - northeast China. Others - the ARGs conferring
resistance to other antibiotic classes, including bacitracin, fosfomycin, imipenem, lantibiotic,
nitroimidazole, pyrazinamide, triclosan, trimethoprim and unknown.
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Fig. S5 Spearman correlations showing the relationships between the number of sites which an
antibiotic resistance gene was detected and log10 transformed absolute abundance (the number of
ARGs per litre water) (A) or log10 transformed normalized abundance (ARG/16S rRNA gene copy
number) of that gene (B). Each black dot indicates an antibiotic resistance gene.
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Fig. S6. Network analysis revealing the co-occurrence patterns between ARGs and bacterial families. The nodes were coloured according to ARG types and bacterial family (n = 41, see Materials and methods).
S8
Table S1 Sample information of 42 lakes and reservoirs for this study Lake/reservoir name Region Lat. (°N) Long. (°E) Province Hubian R. FJ 24.50 118.15 Fujian Bantou R. FJ 24.67 118.02 Fujian Shidou R. FJ 24.69 118.01 Fujian Tingxi R. FJ 24.80 118.14 Fujian Dongzhen R. FJ 25.48 118.94 Fujian Poyang L. CJ 28.99 116.21 Jiangxi Longgan L. CJ 29.94 116.17 Hubei & Anhui Taibai L. CJ 29.96 115.80 Hubei Liangzi L. CJ 30.24 114.51 Hubei Shengjin L. CJ 30.39 117.04 Anhui Nanyi L. CJ 31.12 118.98 Anhui Taihu L. CJ 31.22 120.14 Jiangsu, Zhejiang and
Shanghai Gucheng L. CJ 31.28 118.92 Jiangsu Shijiu L. CJ 31.47 118.89 Anhui & Jiangsu Hongze L. ECC 33.28 118.73 Jiangsu Luoma L. ECC 34.05 118.22 Jiangsu Weishan L. ECC 34.64 117.28 Shandong & Jiangsu Dongping L. ECC 35.97 116.19 Shandong Hengshui L. ECC 37.62 115.63 Heibei Baiyangdian L. ECC 38.94 115.98 Heibei Yuehai L. IM 38.56 106.20 Ningxia Shahu L. IM 38.83 106.36 Ningxia Xinhai L. IM 38.99 106.40 Ningxia Daihai L. IM 40.57 112.67 Inner Mongolia Hasuhai L. IM 40.61 110.97 Inner Mongolia Donghaizi L. IM 40.63 107.00 Inner Mongolia Wuliangsuhai L. IM 40.87 108.79 Inner Mongolia Quansanhaizi L. IM 41.07 107.87 Inner Mongolia Shenglihaizi L. IM 41.12 107.83 Inner Mongolia Bei’er L. IM 47.93 117.70 Inner Mongolia Wulanpao L. IM 48.36 117.52 Inner Mongolia Hulun L. IM 49.12 117.54 Inner Mongolia Huhenuo’er L. IM 49.30 119.23 Inner Mongolia Xinmiaopao L. NEC 45.21 124.45 Jiling Kulipao L. NEC 45.37 124.50 Jiling Xinhuangpao L. NEC 45.63 123.76 Jiling Yueliangpao L. NEC 45.74 124.00 Jiling Lamasipao L. NEC 46.29 124.10 Heilongjiang Amutapao L. NEC 46.61 124.06 Heilongjiang Dongxintunnanpao L. NEC 46.81 124.26 Heilongjiang Qijiapao L. NEC 46.82 124.28 Heilongjiang Tianhu L. NEC 46.87 124.40 Heilongjiang
FJ (included 5 reservoirs) - Fujian province, southeast China; CJ (9 lakes) - the lower and middle reaches of Changjiang River, China; ECC (6 lakes) - east central China, IM (13 lakes) - Inner Mongolia, north China; NEC (9 lakes) - northeast China.
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Table S2 Primer sets used in this study and their target classification (Su, et al., 2015; Zhu, et al., 2013) Gene name Forward primer Reverse primer Classification Mechanism ARDB CARD Other Source
16S rRNA GGGTTGCGCTCGTTGC ATGGYTGTCGTCAGCTCGTG na na
1 - For statistical analyses, we classified these ARGs as efflux pump mechanism; 2 - we classified this ARG as antibiotic deactivate mechanism. FCA -
fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol; MLSB - macrolide, lincosamide and streptogramin; Others - the ARGs conferring
resistance to other antibiotic classes, including bacitracin, fosfomycin, imipenem, lantibiotic, nitroimidazole, pyrazinamide, triclosan, trimethoprim and unknown.
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Explanation of total population, GDP and meat production used as anthropogenic factors in this study
Table S3 Total population, GDP and meat production data collected according to the administrative regions in every basin
Basin Total population × 104 person
GDP × 108 yuan
Meat production × 104 t
Basin area km2
Reference
Hubian R. 97.1 709.19 N/A 73.77 Fujian Provincial Bureau of Statistics, 2013
Bantou R. 60.7 369.26 0.488 275.79
Shidou R. 60.7 369.26 0.488 275.79
Tingxi R. 51.4 198.59 3.2737 657.59
Dongzhen R. 125.4 414.19 7.0852 2324
Poyang L. 4504 12948.88 333.91 166900 Jiangxi Provincial Bureau of Statistics, 2013
Longgan L. 170.45 257.91 10.81 4094.5 Hubei Provincial Bureau of Statistics, 2013; Anhui Provincial Bureau of Statistics, 2013; Susong County Bureau of Statistics, 2013
Taibai L. 151.43 313.1 14.74 2901.35 Hubei Provincial Bureau of Statistics, 2013
Liangzi L. 237.82 1116.93 28.77 5568.8 Hubei Provincial Bureau of Statistics, 2013
Shengjin L. 161.9 417.45 8.4 8271.7 Anhui Provincial Bureau of Statistics, 2013; Chizhou Municipal Bureau of Statistics, 2013
Nanyi L. 121 249.03 5.83 3725.8 Anhui Provincial Bureau of Statistics, 2013; Xuanzhou District Bureau of Statistics, 2013Langxi County Bureau of Statistics, 2013
Taihu L. 5471.95 50916.68 132 37417.4 Jiangsu Provincial Bureau of Statistics, 2013; Zhejiang Statistical Yearbook 2013; National Bureau of Statistics of China, 2013
Gucheng L. 41.91 365.27 1.67 802.8 Jiangsu Provincial Bureau of Statistics, 2013
Shijiu L. 107.58 583.9 3.46 2420 Jiangsu Provincial Bureau of Statistics, 2013; Anhui Provincial Bureau of Statistics, 2013
Hongze L. 540.59 2088.25 36.05 11151.1 Jiangsu Provincial Bureau of Statistics, 2013
Luoma L. 483.98 1669.26 60.46 7585 Jiangsu Provincial Bureau of Statistics, 2013
Weishan L. 2546.55 9530.13 223.31 33769.6 Jiangsu Provincial Bureau of Statistics, 2013; Shandong Provincial Bureau of Statistics, 2013
Dongping L. 684.24 3178.42 51.27 10007.2 Shandong Provincial Bureau of Statistics, 2013
Hengshui L. 438.93 1011.5 31.02 8815 Hebei Provincial Bureau of Statistics, 2013
Baiyangdian L. 1135.14 2720.9 55.2 22190 Hebei Provincial Bureau of Statistics, 2013
Yuehai L. 204.63 1140.83 5.1 9491 Yinchuan Municipal Bureau of Statistics, 2012
Shahu L. 74.16 409.21 2.02 5309.5 Shizuishan Municipal Bureau of Statistics, 2012 Xinhai L. 74.16 409.21 2.02 5309.5
Daihai L. 24.55 74.49 2.29 2494 Inner Mongolia Autonomous Region Bureau of Statistics, 2013 Hasuhai L. 35.98 213.14 2.57 2712
Donghaizi L. 12.29 51.31 0.92 4166.6
Wuliangsuhai L. 34.31 122.92 3.43 7476
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Basin Total population × 104 person
GDP × 108 yuan
Meat production × 104 t
Basin area km2
Reference
Quansanhaizi L. 29.48 98.49 4.15 2492.9
Shenglihaizi L. 29.48 98.49 4.15 2492.9
Bei’er L. 24.75 274.67 4.08 47926
Wulanpao L. 24.75 274.67 4.08 47926
Hulun L. 24.75 274.67 4.08 47926
Huhenuo’er L. 5.86 78.1 1.79 21192
Xinmiaopao L. 289.8 1605.4 36.1 22000 Songyuan Municipal Bureau of Statistics, 2013; Jiling Provincial Bureau of Statistics, 2013
Kulipao L. 289.8 1605.4 36.1 22000
Yueliangpao L. 200.02 615.4 11.4 25685 Baicheng Municipal Bureau of Statistics, 2013; Jiling Provincial Bureau of Statistics, 2013
Xinhuangpao L. 200.02 615.4 11.4 25685
Lamasipao L. 281.7 4000.5 33.8 22161 Daqing Municipal Bureau of Statistics, 2012
Amutapao L. 281.7 4000.5 33.8 22161
Qijiapao L. 281.7 4000.5 33.8 22161
Tianhu L. 281.7 4000.5 33.8 22161
Dongxintunnanpao L. 281.7 4000.5 33.8 22161
Notes:
1. The administrative regions for data collection and estimation of every basin
The catchment descriptors were mainly determined by the principle that if only minor part of an
administrative region is located in a basin, the data of this region wouldn’t be considered in data
collection and estimation for this basin, except that the main cities of this region are located in
the basin; if all or major part of an administrative region is located in a basin, the data of this
region were collected and estimated for this basin. The following are the detailed regions
considered ascovering areas of each basin.
Hubian R.: Huli district in Xiamen prefecture of Fujian province.
Bantou R.: Jimei district in Xiamen prefecture of Fujian province.
Shidou R.: Jimei district in Xiamen prefecture of Fujian province.
Tingxi R.: Tongan district in Xiamen prefecture of Fujian province.
Dongzhen R.: Chenxiang district and Xianyou county in Putian prefecture of Fujian province.
Poyang L.: Jiangxi province
Longgan L.: Huangmei county in Huanggang prefecture of Hubei province; Susong county in
Anqing prefecture of Anhui province.
Taibai L.: Wuxue county and Huangmei county in Huanggang prefecture of Hubei province.
Liangzi L.: Jiangxia district in Wuhan prefecture of Hubei province; Liangzihu district in Ezhou
prefecture of Hubei province; Xianan district in Xianning prefecture of Hubei province; Daye
county in Huangshi prefecture of Hubei province.
Shengjin L.: Chizhou prefecture of Anhui province.
Nanyi L.: Xuanzhou district and Langxi county in Xuancheng prefecture of Anhui province.
Taihu L.: Wuxi, Changzhou, Zhengjiang and Suzhou prefectures of Jiangsu province; Shanghai;
Huzhou and Jiaxin prefectures of Zhejiang province.
Gucheng L.: Gaochun district in Nanjing prefecture of Jiangsu province.
Shijiu L.: Lishui district in Nanjing prefecture of Jiangsu province;
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Hongze L.: Huaian prefecture excluding counties of Lianshui and Jinhu; Sihong and Siyang
counties in Suqian prefecture of Jiangsu province.
Luoma L.: Suqian prefecture excluding counties of Sihong, Siyang and Muyang; Xinyi, Pizhou
and Suining counties in Xuzhou prefecture of Jiangsu province.
Weishan L.: Xuzhou prefecture excluding counties of Xinyi, Pizhou and Suining; Jining,
Zaozhuang and Heze prefectures of Shandong province.
Dongping L.: Taian and Laiwu prefectures of Shandong province.
Hengshui L.: Hengshui prefecture of Hebei province.
Baiyangdian L.: Baoding prefecture of Hebei province.
Yuehai L.: Yingchuan prefecture of Ningxia Hui Autonomous Region.
Shahu L.: Shizuishan prefecture of Ningxia Hui Autonomous Region.
Xinhai L.: Shizuishan prefecture of Ningxia Hui Autonomous Region.
Daihai L.: Liangcheng county in Ulanqab prefecture of Inner Mongolia Autonomous Region.
Hasuhai L.: Tumote left banner in Hohhot prefecture of Inner Mongolia Autonomous Region.
Donghaizi L.: Dengkou county in Bayannaoer prefecture of Inner Mongolia Autonomous
Region
Wuliangsuhai L.: Wulate Front Banner in Bayannaoer prefecture of Inner Mongolia
Autonomous Region.
Quansanhaizi L.: Wuyuan county in Bayannaoer prefecture of Inner Mongolia Autonomous
Region.
Shenglihaizi L.: Wuyuan county in Bayannaoer prefecture of Inner Mongolia Autonomous
Region.
Bei’er L.: Xinbaerhu right banner, Xinbaerhu left banner and Manzhouli county in Hulunbuir
prefecture of Inner Mongolia Autonomous Region.
Wulanpao L.: Xinbaerhu right banner, Xinbaerhu left banner and Manzhouli county in
Hulunbuir prefecture of Inner Mongolia Autonomous Region.
Hulun L.: Xinbaerhu right banner, Xinbaerhu left banner and Manzhouli county in Hulunbuir
prefecture of Inner Mongolia Autonomous Region.
Huhenuo’er L.: Chenbaerhu Banner in Hulunbuir prefecture of Inner Mongolia Autonomous
Region.
Xinmiaopao L.: Songyuan prefecture of Jiling province.
Kulipao L.: Songyuan prefecture of Jiling province.
Yueliangpao L.: Baicheng prefecture of Jiling province.
Xinhuangpao L.: Baicheng prefecture of Jiling province.
Lamasipao L.: Daqing prefecture of Heilongjiang province.
Amutapao L.: Daqing prefecture of Heilongjiang province.
Qijiapao L.: Daqing prefecture of Heilongjiang province.
Tianhu L.: Daqing prefecture of Heilongjiang province.
Dongxintunnanpao L.: Daqing prefecture of Heilongjiang province.
2. General Description of indicators
2.1. All data of indicators for each basin are derived from statistical data of the administrative
regions where were mainly covered by each basin, if not, the mainpopulation or larger cities in
administrative regions should be distributed within thebasin.
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2.2. Administrative region population is estimated in according with the annual sample survey on
population changes.
2.3. Meat production is the sum of pork, beef, poultry, mutton, and rabbit meat productions.
2.4. All the administrative region area used in this study is according to the latest administrative
division.
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Explanation of municipal domestic sewage factor
Table S4 Municipal domestic sewage data estimated according to the administrative regions in every basin Basin Municipal domestic sewage × 106 t Reference
Hubian R. 38.78
China Environment Year Book Editorial Board, 2013
Bantou R. 24.24
Shidou R. 24.24
Tingxi R. 20.53
Dongzhen R. 50.09
Poyang L. 1331.00
Longgan L. 55.89
Taibai L. 51.96
Liangzi L. 81.61
Shengjin L. 50.56
Nanyi L. 37.79
Taihu L. 3131.82
Gucheng L. 19.15
Shijiu L. 39.65
Hongze L. 246.95
Luoma L. 221.09
Weishan L. 901.81
Dongping L. 208.49
Hengshui L. 110.21
Baiyangdian L. 285.03
Yuehai L. 70.85
Shahu L. 25.68
Xinhai L. 25.68
Daihai L. 6.78
Hasuhai L. 9.94
Donghaizi L. 3.40
Wuliangsuhai L. 9.48
Quansanhaizi L. 8.15
Shenglihaizi L. 8.15
Bei’er L. 6.84
Wulanpao L. 6.84
Hulun L. 6.84
Huhenuo’er L. 1.62
Xinmiaopao L. 78.61
Kulipao L. 78.61
Yueliangpao L. 54.26
Xinhuangpao L. 54.26
Lamasipao L. 76.56
Amutapao L. 76.56
Qijiapao L. 76.56
Tianhu L. 76.56
Dongxintunnanpao L. 76.56
Note:
Municipal domestic sewage effluent (S) is mainly estimated according to the following equation.
S = So × P/Po (1)
Where So, municipal domestic sewage effluent of province; P, population in the administrative
regions in Table S3; Po, population in the province in Table S2.
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Explanation of aquatic production, number of patient diagnosed and treated, number of residential patient factors
Table S5 Aquatic production, number of patient diagnosed and treated, number of residential patient data in 2012 Basin Aquatic
production × 104 t
No. of patients diagnosed and treated × 108 person-time
No. of residential patients × 106 person-time
Reference
Hubian R. 82.08 1.92 510.16
National Bureau of Statistics of China, 2013
Bantou R. 82.08 1.92 510.16
Shidou R. 82.08 1.92 510.16
Tingxi R. 82.08 1.92 510.16
Dongzhen R. 82.08 1.92 510.16
Poyang L. 237 1.9 657.87
Longgan L. 596.44 5.41 1573.73
Taibai L. 388.95 3.06 862.08
Liangzi L. 388.95 3.06 862.08
Shengjin L. 207.49 2.35 711.65
Nanyi L. 207.49 2.35 711.65
Taihu L. 470.25 11.24 1855.98
Gucheng L. 345.26 4.51 952.9
Shijiu L. 552.75 6.86 1664.55
Hongze L. 345.26 4.51 952.9
Luoma L. 345.26 4.51 952.9
Weishan L. 501.08 10.34 2350.38
Dongping L. 155.82 5.83 1397.48
Hengshui L. 52.85 3.68 869.6
Baiyangdian L. 52.85 3.68 869.6
Yuehai L. 12.35 0.31 82
Shahu L. 12.35 0.31 82
Xinhai L. 12.35 0.31 82
Daihai L. 13.16 0.93 258.63
Hasuhai L. 13.16 0.93 258.63
Donghaizi L. 13.16 0.93 258.63
Wuliangsuhai L. 13.16 0.93 258.63
Quansanhaizi L. 13.16 0.93 258.63
Shenglihaizi L. 13.16 0.93 258.63
Bei’er L. 13.16 0.93 258.63
Wulanpao L. 13.16 0.93 258.63
Hulun L. 13.16 0.93 258.63
Huhenuo’er L. 13.16 0.93 258.63
Xinmiaopao L. 18.21 0.97 302.45
Kulipao L. 18.21 0.97 302.45
Yueliangpao L. 18.21 0.97 302.45
Xinhuangpao L. 18.21 0.97 302.45
Lamasipao L. 45.28 1.15 420.09
Amutapao L. 45.28 1.15 420.09
Qijiapao L. 45.28 1.15 420.09
Tianhu L. 45.28 1.15 420.09
Dongxintunnanpao L. 45.28 1.15 420.09
Note:
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The data of aquatic production, number of patient diagnosed and treated and number of
residential patient refer to the corresponding values in the province where each basin is located.
Number of patient diagnosed and/or treated and number of residential patient are sourced from
China Statistical Yearbook 2013 (National Bureau of Statistics of China, 2013). Aquatic
production includes aquaculture and capture in freshwater, but the latter only accounts for a
small proportion of total production in most basins.
S28
Table S6 ARG groups in the south/central China compared with the north Chinese lakes/reservoirs (independent sample t-test)
Absolute abundance (P) Normalized abundance (P)
Aminoglycoside < 0.05 < 0.05
Beta-Lactamase > 0.05 > 0.05
FCA > 0.05 > 0.05
MLSB > 0.05 > 0.05
Multidrug > 0.05 < 0.05
Sulfonamide > 0.05 > 0.05
Tetracycline > 0.05 > 0.05
Vancomycin > 0.05 > 0.05
Others > 0.05 > 0.05
All ARGs > 0.05 < 0.01
FCA - fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol; MLSB -
macrolide, lincosamide and streptogramin; Others - the ARGs conferring resistance to other
antibiotic classes, including bacitracin, fosfomycin, imipenem, lantibiotic, nitroimidazole,
pyrazinamide, triclosan, trimethoprim and unknown.
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Table S7 The richness and absolute abundance of ARGs that are unique and shared between the south/central and the north Chinese lakes/reservoirs