Prevalence of antibiotic resistance genes in antibiotic resistantEscherichia coli isolates in surface water of Taihu Lake Basin, China
Songhe Zhanga, b,*, Xiaoyang, Lva, Bing Hana, Xiucong Gua,, Peifang Wanga ,*, Chao Wanga, Zhenli Heb
a Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes of Ministry of Education, College of Environment, Hohai University, Xikang Road No. 1, Gulou District, Nanjing 210098, China.
b University of Florida. Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945, USA.
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Methods and materials.
To isolate the E. coli from food animal and human source, three regions were established for sampling. Effluent samplesof biological treated wastewater from chicken and swine feedlots and from wastewater treatment plant were collected from each region (May, 2010).The wastewater treatment plantsreceived and treated about 700,000-1,500,000 ton of wastewater every day. About 30,000--50, 000 chickens and 3,0000-50000 pigs were produced in the chicken and swine feedlots annually.
A total of 21 TET-resistance genes (Aminovet al. 2001, 2002; Ng et al. 2001), 2 streptomycin-resistance genes (Dalsgaard et al., 1999; Hochhut et al. 2001), 4 sulfonamide-resistance gene (Pei et al. 2006), 8 β–lactamases genes (Ahmed et al. 2007; Henriques et al. 2006) and 4 quinolone-resistance genes (Robicsek et al. 2006) were detected in these isolates. The presence of class I integron was also detected according to the reference (Mazel 2000).The selected clones were cultured in LB liquid medium at 37 ºC for 8 h, then 200 μL cultures were collected and centrifuged at 5, 000 xg for 5 min. After washed with double distilled H2O (ddH2O) twice, the pellets were re-suspended in 50 μL ddH2O. The suspended solution were heated at 95 ºC for 10 min and centrifuged at 12,000 xg for 5 min. The supernatant solution was used as templates for PCR amplification.
PCR assays were carried out using a Takara Ex Taq kit (Takara) in a 25-μL volume reaction. The PCR mixture consisted of 2.5 μL of 10× Ex Taq buffer (Mg2+Plus), 2 μL of dNTPs (2.5 mM each), 0.1 μM of each primer, 2 μL of E. coli DNA, and 0.15 μL 5 U/μL Ex Taq DNA polymerase. Each PCR was performed with PTC-200 (Bio-rad). The PCR was initiated by incubating the reaction mixture at 95 ºC for 4 min, followed by 40 cycles of 30 s at 95 ºC for denaturation, 30 s at the annealing temperature (for the annealing temperatures, see Supporting Information Table S2), and 45 s at 72 ºC for extension.
To generate positive controls, purified PCR products obtained from the resistant isolates were cloned and sequenced. If the PCR products were verified as the object resistance genes using the BLAST alignment tool ( these isolates with such genes were used as the positive control. E. coli ATCC 25922 and all the reagents without any template were used as negative controls.Both positive and negative controls were included in each run. PCR products were verified by 1.0% (w/v) agarose gel electrophoresis.
Figure. S1. Map of sampling points in Lake Taihu (a-h#) and its seventeen tributaries 1-17#).●, Surface water sampling sites.,Sampling sites with WWTPs,swine andchicken sourses;*,E. garzetta source.
FigureS2. Neighbor-joining phylogenetic analysis of qnrB diversity in E. coli isolated from Taihu lake basin. Acc No. HE575214-HE575220 were submitted in this study.
TableS1 Sampling sites and concentration of faecal coliforms (CFU/L)Water body Sites / Locations / GPS information / Inlet/ outlet / Faecal coliforms
N; E / May 2010 / Dec 2009
Tahhu Lake / a# / Meiliang Bay / 31°29'972"; 120°09'738" / \ / 490(26) / 240(21)
b# / Zhushan Bay / 31°25'404"; 120°02'871" / \ / 340(25) / 270(22)
c# / Xibuyanan / 31°18'145"; 119°58'350" / \ / 693(11) / 320 (15)
d# / Huxin / 31°16'046"; 120°07'197" / \ / 245(12) / 105(8)
e# / Nanbu / 31°04'751"; 120°01'785" / \ / 320(9) / 115(7)
f# / Nanbu Bay / 30°57'196"; 120°14'493" / \ / 420(17) / 240(11)
g# / Dongbu Bay / 31°10'905"; 120°25'708" / \ / 465(7) / 144(12)
h# / Gonghu Bay / 31°29'575"; 120°12'442" / \ / 315(15) / 210(8)
River / 1# / Wangyu / 31°27'5"; 120°25'10" / inlet / 1030(94) / \
2# / Li / 31°28'46″; 120°20'46″ / inlet / 3730(135)* / \
3# / Changguang / 31°27'25″; 120°16'3″ / inlet / 1810(73) / \
4# / liangxi / 31°33'2″; 120°13'44″ / inlet / 2530(175)* / \
5# / zhihugang / 31°33'35″; 120°7'31″ / inlet / 1740(65) / \
6# / Taige canal / 31°31'10″; 120°0'54″ / inlet / 2230(79)* / \
7# / Shaoxiang / 31°25'38″; 119°58'45″ / inlet / 1480(44) / \
8# / Guandugang / 31°20'14″; 119°56'15″ / inlet / 1740(102) / \
9# / Chendonggang / 31°19'22″; 119°55'48″ / inlet / 1360(64) / \
10# / Zhudugang / 31°17'13″; 119°54'29″ / inlet / 2210(24)* / \
11# / Jiapugang / 31°6'15″; 119°56'12″ / inlet / 1450(21) / \
12# / Changxinggang / 30°0'7″; 119°54'39″ / inlet / 1445(27) / \
13# / Dongtiaoxi / 31°50'57″; 120°5'38″ / inlet / 1960(157) / \
14# / Dapukou / 30°59'49″; 120°38'16″ / inlet / 2270(112)* / \
15# / Taipu / 31°11'52″; 120°38'41″ / outlet / 814(47) / \
16# / Wusongjiang / 31°17'56″; 120°23'7″ / outlet / 1210(76) / \
17# / Huguang canal / 31°13'20″; 120°27'59″ / outlet / 1970(74) / \
*Fecal coliform concentration is above the limits set by the World Health Organization for recreational water (200 fecal coliforms per 100 ml) ; Numbers inside thebrackets indicate the standard errors.
Table S2. PCRPrimers used in this study.
primers / Target / Sequence (5`-3`) / Annealing temp (°C) / Amplicon Size (bp) / Referencetet(A)-FW / tet(A) / GCTACATCCTGCTTGCCTTC / 60 / 210 / Ng et al. 2001
tet(A)-RV / CATAGATCGCCGTGAAGAGG
tet(B)-FW / tet(B) / TTGGTTAGGGGCAAGTTTTG / 58 / 659
tet(B)-RV / GTAATGGGCCAATAACACCG
tet(C)-FW / tet(C) / CTTGAGAGCCTTCAACCCAG / 62 / 418
tet(C)-RV / ATGGTCGTCATCTACCTGCC
tet(D)-FW / tet(D) / AAACCATTACGGCATTCTGC / 62 / 787
tet(D)-RV / GACCGGATACACCATCCATC
tet(E)-FW / tet(E) / AAACCACATCCTCCATACGC / 60 / 278
tet(E)-RV / AAATAGGCCACAACCGTCAG
tet(G)-FW / tet(G) / CAGCTTTCGGATTCTTACGG / 62 / 844
tet(G)-RV / GATTGGTGAGGCTCGTTAGC
tet(K)-FW / tet(K) / TCGATAGGAACAGCAGTA / 54 / 169
tet(K)-RV / CAGCAGATCCTACTCCTT
tet(L)-FW / tet(L) / TCGTTAGCGTGCTGTCATTC / 45 / 267
tet(L)-RV / GTATCCCACCAATGTAGCCG
tet(M)-FW / tet(M) / GTGGACAAAGGTACAACGAG / 60 / 406
tet(M)-RV / CGGTAAAGTTCGTCACACAC
tet(O)-FW / tet(O) / AACTTAGGCATTCTGGCTCAC / 45 / 515
tet(O)-RV / TCCCACTGTTCCATATCGTCA
tet(S)-FW / tet(S) / CATAGACAAGCCGTTGACC / 58 / 667
tet(S)-RV / ATGTTTTTGGAACGCCAGAG
tetA(P)-FW / tetA(P) / CTTGGATTGCGGAAGAAGAG / 58 / 676
tetA(P)-RV / ATATGCCCATTTAACCACGC
tet(Q)-FW / tet(Q) / TTATACTTCCTCCGGCATCG / 60 / 904
tet(Q)-RV / ATCGGTTCGAGAATGTCCAC
tet(X)-FW / tet(X) / CAATAATTGGTGGTGGACCC / 54 / 468
tet(X)-RV / TTCTTACCTTGGACATCCCG
tet(30)-FW / tet(30) / CATCTTGGTCGAGGTGACTGG / 45 / 134 / Aminov et al., 2001
tet(30)-RV / ACGAGCACCCAGCCGAGC
tet(H)-FW / tet(H) / CAGTGAAAATTCACTGGCAAC / 58 / 185
tet(H)-RV / ATCCAAAGTGTGGTTGAGAAT
tet(J)-FW / tet(J) / CGAAAACAGACTCGCCAATC / 55 / 184
tet(J)-RV / TCCATAATGAGGTGGGGC
tet(T)-FW / tet(T) / AAGGTTTATTATATAAAAGTG / 45 / 169 / Aminov et al., 2002
tet(T)-RV / AGGTGTATCTATGATATTTAC
tet(W)-FW / tet(W) / GAGAGCCTGCTATATGCCAGC / 45 / 168
tet(W)-RV / AGGTGTATCTATGATATTTAC
tet(Y)-FW / tet(Y) / ATTTGTACCGGCAGAGCAAAC / 45 / 181
tet(Y)-RV / GGCGCTGCCGCCATTATGC
tet(Z)-FW / tet(Z) / CCTTCTCGACCAGGTCGG / 55 / 204
tet(Z)-RV / ACCCACAGCGTGTCCGTC
sul(I)-FW / sul(I) / CGCACCGGAAACATCGCTGCAC / 45 / 163 / Pei et al. 2006
sul(I)-RV / TGAAGTTCCGCCGCAAGGCTCG
sul(II)-FW / sul(II) / TCCGGTGGAGGCCGGTATCTGG / 45 / 191
sul(II)-RV / CGGGAATGCCATCTGCCTTGAG
sul(III)-FW / sul(III) / TCCGTTCAGCGAATTGGTGCAG / 45 / 128
sul(III)-RV / TTCGTTCACGCCTTACACCAGC
sul(A)-FW / sul(A) / TCTTGAGCAAGCACTCCAGCAG / 45 / 299
sul(A)-RV / TCCAGCCTTAGCAACCACATGG
CARB-F / CARB / CAAGTACTTTYAAAACAATAGC / 46 / 534 / Henriques et al. 2006
CARB-R / GCTGTAATACTCCKAGCAC
blaSHV-FW / blaSHV / GCGAAAGCCAGCTGTCGGGC / 45 / 304
blaSHV-RV / GATTGGCGGCGCTGTTATCGC
blaTEM-FW / blaTEM / AAAGATGCTGAAGATCA / 45 / 425
blaTEM-RV / TTTGGTATGGCTTCATTC
blaCTX-M-F / blaCTX-M / CGCTTTGCGATGTGCAG / 51 / 550 / Ahmed et al. 2007
blaCTX-M-R / ACCGCGATATCGTTGGT
blaCMY-F / blaCMY / GACAGCCTCTTTCTCCACA / 55 / 1015
blaCMY-R / TGGAACGAAGGCTACGTA
blaOXA-F / blaOXA / TCAACTTTCAAGATCGCA / 45 / 591
blaOXA-R / GTGTGTTTAGAATGGTGA
blaOxy-F / blaOxy / GGTTTTGGTAACTGTGACGGG / 45 / 725
blaOxy-R / CAGAGTGCAGAGTGTTGCAG
NDM-FW / NDM-1 / GGCGGAATGGCTCATCACGA / 58 / 286 / Chen et al. 2011
NDM-RV / CGCAACACAGCCTGACTTTC
qnrA-FW / qnr(A) / ATTTCTCACGCCAGGATTTG / 54 / 516 / Robicsek et al. 2006
qnrA-RV / GATCGGCAAAGGTTAGGTCA
qnrB-FW / qnr(B) / GATCGTGAAAGCCAGAAAGG / 56 / 469
qnrB-RV / ACGATGCCTGGTAGTTGTCC
qnrS-FW / qnr(S) / ACGACATTCGTCAACTGCAA / 54 / 417
qnrS-RV / TAAATTGGCACCCTGTAGGC
aac(6`)-Ib-F / aac(6`)-Ib / TTGCGATGCTCTATGAGTGGCTA / 50 / 482
aac(6`)-Ib-R / CTCGAATGCCTGGCGTGTTT
StrA-FW / Str(A) / TTGATGTGGTGTCCCGCAATGC / 57 / 383 / Hochhut et al. 2001
StrA -RV / CCAATCGCAGATAGAAGGCAA
StrB-FW / Str(B) / CCGCGATAGCTAGATCGCGTT / 48 / 470 / Ramachandran et al. 2007
StrS-RV / CGACTACCAGGCGACCGAAAT
Int-1F / Int I / GGGTCAAGGATCTGGATTTCG / Mazel et al. 2000
Int-1r / ACATGCGTGTAAATCATCGTCG
5`CS / gene cascades of Int-1 / GGCATCCAAGCAGCAAG / variable / Lévesque et al. 1995
3`CS / AAGCAGACTTGACCTGA
BOX-A1R / CTACGGCAAGGCGACGCTGACG / Versalovic et al . 1994
Table S3.Characterization of antibiotic resistantE. coliisolated from surface water of Taihu Lake and tributary rivers.
Site / TET / OTC / GT / AM / SM / LE / NA / TS / CF / Total / MAR indexTaihu, Dec, 2009 / #a / 5 / 5 / 6 / 8 / 5 / 3 / 5 / 7 / 6 / 15 / 0.37
#b / 9 / 2 / 8 / 3 / 6 / 1 / 3 / 11 / 5 / 15 / 0.36
#c / 4 / 3 / 3 / 7 / 4 / 1 / 8 / 8 / 6 / 15 / 0.33
#d / 7 / 4 / 6 / 5 / 7 / 2 / 3 / 8 / 5 / 15 / 0.35
#e / 4 / 5 / 8 / 7 / 5 / 1 / 9 / 11 / 4 / 15 / 0.4
#f / 6 / 5 / 4 / 3 / 5 / 4 / 7 / 6 / 4 / 15 / 0.33
#g / 3 / 8 / 7 / 11 / 6 / 4 / 11 / 10 / 6 / 15 / 0.49
#h / 7 / 2 / 7 / 8 / 6 / 3 / 6 / 12 / 7 / 15 / 0.43
χ2value (v=7) / 7.93 / 9.03 / 6.31 / 14.12* / 1.72 / 5.94 / 15.20* / 9.2 / 2.28 / / / /
sub-total / 45 / 34 / 49 / 52 / 44 / 19 / 52 / 73 / 43 / 120
Taihu May, 2010 / #a / 9 / 4 / 6 / 4 / 7 / 4 / 7 / 7 / 6 / 15 / 0.4
#b / 8 / 5 / 8 / 6 / 2 / 3 / 6 / 11 / 7 / 15 / 0.41
#c / 8 / 5 / 7 / 7 / 1 / 4 / 5 / 9 / 5 / 15 / 0.38
#d / 9 / 4 / 2 / 8 / 5 / 2 / 4 / 8 / 6 / 15 / 0.36
#e / 5 / 4 / 5 / 7 / 3 / 1 / 9 / 11 / 5 / 15 / 0.37
#f / 8 / 4 / 7 / 2 / 4 / 4 / 5 / 9 / 7 / 15 / 0.37
#g / 9 / 6 / 5 / 5 / 5 / 3 / 6 / 11 / 8 / 15 / 0.43
#h / 6 / 7 / 8 / 5 / 3 / 5 / 4 / 7 / 6 / 15 / 0.38
χ2value(v=7) / 14.68* / 2.7 / 7.78 / 7.46 / 9.07 / 4.52 / 5.5 / 5.84 / 2.06 / / / /
sub-total / 62 / 39 / 48 / 44 / 30 / 26 / 46 / 73 / 50 / 120
Taihu total x2 value (v=15) / 16.58 / 11.95 / 14.1 / 22.78 / 3.68 / 11.57 / 21.45 / 15.04 / 518 / /
River / 1# / 5 / 6 / 11 / 14 / 10 / 8 / 7 / 15 / 11 / 25 / 0.64
2# / 12 / 18 / 13 / 14 / 11 / 18 / 15 / 16 / 6 / 25 / 0.55
3# / 11 / 8 / 10 / 8 / 6 / 4 / 10 / 17 / 15 / 25 / 0.4
4# / 8 / 13 / 13 / 15 / 9 / 1 / 15 / 17 / 10 / 25 / 0.45
5# / 10 / 6 / 7 / 5 / 9 / 4 / 8 / 17 / 8 / 25 / 0.33
6# / 13 / 10 / 10 / 9 / 5 / 0 / 12 / 18 / 14 / 25 / 0.4
7# / 15 / 11 / 7 / 4 / 6 / 7 / 7 / 16 / 9 / 25 / 0.36
8# / 16 / 7 / 12 / 5 / 8 / 8 / 10 / 19 / 11 / 25 / 0.43
9# / 11 / 16 / 11 / 8 / 7 / 3 / 18 / 21 / 7 / 25 / 0.45
10# / 9 / 15 / 12 / 12 / 6 / 6 / 17 / 16 / 9 / 25 / 0.45
11# / 12 / 13 / 10 / 11 / 10 / 7 / 8 / 15 / 8 / 25 / 0.42
12# / 15 / 13 / 16 / 20 / 15 / 5 / 14 / 24 / 10 / 25 / 0.59
13# / 8 / 11 / 11 / 13 / 12 / 3 / 18 / 21 / 12 / 25 / 0.48
14# / 3 / 14 / 22 / 9 / 4 / 5 / 12 / 22 / 10 / 25 / 0.45
15# / 6 / 8 / 11 / 9 / 7 / 9 / 7 / 16 / 5 / 25 / 0.35
16# / 8 / 8 / 6 / 7 / 4 / 3 / 15 / 17 / 8 / 25 / 0.34
17# / 15 / 11 / 12 / 16 / 0 / 2 / 7 / 16 / 6 / 25 / 0.38
χ2value (v=16) / 37.88* / 33.23* / 5.38 / 50.57* / 37.87* / 63.70* / 42.88* / 21.98 / 20.48
sub-total / 177 / 188 / 194 / 179 / 129 / 93 / 200 / 303 / 159 / 425
Total number of resistance isolates
from surface water (percentages) / 284
(42.71%) / 261
(39.25%) / 291
(43.76%) / 275
(41.35%) / 203
(30.53%) / 138
(20.75%) / 298
(44.81%) / 449
(67.52%) / 252
(37.89%) / 665 / 0.41
*indicates significant difference between groups (P<0.05)
Table S4. Distribution of E. coli Resistant to Antibiotics from human, feedlots and wildlife sources.
TET / OTC / GT / AM / SM / LE / NA / TS / CF / Total / MAR indexChicken / 17 / 16 / 20 / 19 / 15 / 9 / 22 / 20 / 17 / 27 / 0.64
Swine / 21 / 14 / 20 / 17 / 18 / 13 / 18 / 18 / 10 / 29 / 0.57
Human / 20 / 11 / 12 / 15 / 12 / 3 / 13 / 22 / 13 / 45 / 0.3
E. garzetta / 2 / 5 / 4 / 3 / 2 / 2 / 5 / 7 / 3 / 15 / 0.15
χ2value (v=3)§ / 16.15* / 9.84* / 23.39* / 15.26* / 16.30* / 16.85* / 22.08* / 5.37 / 11.01*
*indicates significant difference between groups (P<0.05). §χ2values were calculated using the percentages of the ARBs in the sample.
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