Supplementary Materials: Abundances of Clinically Relevant ... - MDPI

Supplementary Materials: Abundances of Clinically Relevant Antibiotic Resistance Genes and Bacterial Community Diversity in the Weihe River, China Xiaojuan Wang 1, Jie Gu 1, *, Hua Gao1, Xun Qian1 and Haichao Li 1

Figure S1. Total relative abundances of ARGs and intI1 in water samples collected from the Weihe River.

Figure S2. Venn diagrams showing the shared and distinct operational taxonomic units (OTUs) in different sites.

Int. J. Environ. Res. Public Health 2018, 15

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Figure S3. Distributions of the relative abundances of the 10 most abundant bacteria in the 13 samples at different levels: (a) phylum level; (b) class level; (c) order level; and (d) family level.


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Table S1. Descriptions of the sampling sites. City

Baoji

Xianyang Xi’an

Weinan

Sample ID

Site

Urban/Rural

Location

S1

Linjia Village

Rural

107.05E 34.36N

S2

Wolongsi Bridge

Urban

107.27E 34.34N

S3

Guozhen Bridge

Urban

107.39E 34.33N

S4

Changxing Bridge

Rural

107.83E 34.21N

S5

Xingping

Rural

108.51E 34.23N

S6

Nanying

Rural

108.65E 34.28N

Xianyang Railway Bridge

Rural

108.81E 34.37N

Tianjiangrendu

Urban

108.97E 34.40N

S9

Gengzhen Bridge

Urban

109.11E 34.45N

S10

Xinfeng Bridge

Urban

109.26E 34.47N

S11

Shawangdu

Urban

109.50E 34.53N

S12

Shuyuan

Urban

109.60E 34.52N

S13

Tongguan Suspension Bridge Rural

and S7 S8

110.24E 34.62N


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Table S2. Primers and probes for the detection of clinically relevant antibiotic genes in this study. Class -lactam

Target gene blaTEM mecA

vancomycin

vanA

streptomycin

strA strB aadA

intI1 16S rRNA

Primer sequence (5→3) F-CACTATTCTCAGAATGACTTGGT R-TGCATAATTCTCTTACTGTCATG F-CGCAACGTTCAATTTAATTTTGTTAA R-TGGTCTTTCTGCATTCCTGGA F-CTGTGAGGTCGGTTGTGCG R-TTTGGTCCACCTCGCCA F-TCAATCCCGACTTCTTACCG R-CACCATGGCAAACAACCATA F-ATCGCTTTGCAGCTTTGTTT R-ATGATGCAGATCGCCATGTA F-CAGCGCAATGACATTCTTGC R-GTCGGCAGCGACA(C/T)CCTTCG F-GCCTTGATGTTACCCGAGAG R-GATCGGTCGAATGCGTGT F-TCCTACGGGAGGCAGCAGT R-GGACTACCAGGGTATCTAATCCTGTT

Probe sequence FAM-CCAGTCACAGAAAAGCATCTTACGG-BHQ1

Amplicon size (bp) 85

Reference [31]

VIC-AATGACGCTATGATCCCAATCTAACTTCCACA-BHQ1

92

[32]

FAM-CAACTAACGCGGCACTGTTTCCCAAT-BHQ1

64

[33]

FAM-TGCTCGACCAAGAGCGGC-BHQ1

126

VIC-ATGCCTCGGAACTGCGT-BHQ1

143

FAM-TGGTAGGTCCAGCGGCGGAG-BHQ1

295

VIC-ATTCCTGGCCGTGGTTCTGGGTTTT-BHQ1

196

[35]

VIC-CGTATTACCGCGGCTGCTGGCAC-BHQ1

466

[34]

[34]

Table S3. Raw and clean tags, OTUs, Good’s coverage, and Shannon, Chao1, ACE, and Simpson’s indices for the 13 water samples. Sample ID S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

Raw tags 147375 412139 90719 45508 103522 215539 67992 141226 52020 63319 60770 48273 93267

Effective tags 75725 206956 42527 20520 53191 107237 33923 65258 23979 29698 32636 24085 46152

OTU number 1471 1849 1148 974 1270 1554 996 1617 1140 1190 1424 1286 1584

Good’s coverage 0.978 0.98 0.982 0.97 0.98 0.979 0.981 0.975 0.985 0.981 0.976 0.982 0.977

Shannon 6.747 5.488 6.489 6.73 6.37 6.476 6.218 6.995 7.041 6.864 6.616 6.921 7.619

Chao 1 1316.694 1189.152 1067.38 2835.082 1242.435 1290.15 1179.19 1453.173 1092.96 1141.638 1494.455 1234.444 1498.083

ACE 1395.271 1244.642 1127.118 1746.054 1282.704 1303.588 1180.159 1559.622 1132.75 1275.748 1553.858 1314.747 1568.162

Simpson 0.961 0.869 0.967 0.97 0.944 0.953 0.944 0.974 0.976 0.973 0.933 0.951 0.98


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Table S4. Environmental factors used for redundancy analysis. Sample ID

Electrical conductivity (s/cm)

Temperature (°C)

pH

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

744 755 925 829 880 891 874 808 656 975 874 950 885

18.2 20.0 21.5 19.7 18.4 19.7 20.3 14.8 13.2 12.2 13.8 14.8 13.2

7.62 8.57 8.69 8.10 8.86 8.70 8.91 8.18 7.88 7.97 7.85 7.96 7.98

Dissolved oxygen (mg/L) 10.42 3.56 8.82 11.62 9.26 9.63 9.40 10.00 8.45 8.06 6.58 7.67 7.78

Table S5. Pearson’s correlation coefficients between genes and the main bacterial phyla * Significantly different at p < 0.05. **Significantly different at p < 0.01. bla TEM 0.154 0.049 –0.15 –0.070 –0.464 –0.023

Proteobacteria Actinobacteria Cyanobacteria Bacteroidetes Verrucomicrobia Firmicutes

strA 0.04 0.025 –0.073 –0.093 –0.063 0.214

strB 0.575* –0.891** –0.439 0.469 –0.631* 0.574*

aadA 0.089 0.08 –0.26 0.302 –0.341 –0.181

mecA 0.024 0.08 –0.119 0.064 –0.091 –0.064

intI1 0.729** –0.59* –0.670* 0.167 –0.676* 0.534

vanA –0.238 0.038 0.247 0.050 –0.013 0.027

Table S6. Pearson’s correlation coefficients between the relative abundances of ARGs and the intI1 gene. * Significantly different at p < 0.05. blaTEM mecA vanA strA strB aadA intI1

bla TEM 1.000 –0.370 –0.249 –0.443 –0.051 0.515 0.583*

mecA

vanA

strA

strB

aadA

intI1

1.000 0.254 0.534 –0.144 –0.318 –0.381

1.000 0.480 –0.122 –0.296 –0.354

1.000 –0.008 –0.573* –0.468

1.000 –0.051 0.483

1.000 0.640*

1.000

Table S7. Pearson’s correlation coefficients between genes and environmental factors. * Significantly different at p < 0.05. pH Dissolved oxygen Temperature Electrical conductivity

bla TEM –0.425 –0.235 –0.599*

strA –0.348 –0.041 –0.084

strB 0.164 –0.536 0.342

aadA –0.290 0.246 –0.300

mecA –0.238 0.216 –0.188

intI1 –0.141 –0.44 –0.161

vanA –0.497 0.210 0.032

–0.261

–0.063

0.224

–0.121

0.038

–0.335

–0.197

© 2018 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).