Characterization of Antibiotic Resistance Genes in Antibiotic Resistance E. Coli Isolates

Characterization of antibiotic resistance genes in antibiotic resistance E. coli isolates from a Lake

Chao Wang, XiuCong Gu, Songhe Zhang*, Peifang Wang, Chuan Guo, Ju Gu, Jun Hou.

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.

Corresponding Author*: E-mail: or .

Supplemental Information

Materials and Methods

Fig S1. Sampling sites in three sub-lakes JSA (J1-3), JSB (J4) and JSC (J5-J8).

Sampling Sites

Eight surface water samples were collected from the indicated sites in JinSha Lake (Figure S1). The JinSha Lake had been segmented into three parts, JSA, JSB and JSC, for ecological remediation in recent years. There were many villages near sites J1 and J3. Site J2 was influenced by a middle school and J3 was located at the inlets of a river. Site 4 located in JSB, which had been dredged and groundwater was the main water source. The water has ever been affected serious by domestic wastewater at sites J5-8 and now most of residents around these sites have now relocated to other places. The physical and chemical properties of water samples were listed in Table S1.

Table S1 Quality of water in the sampling site of JinSha Lake(means+s.d.).

Location / pH / Total Phosphate
(mg/L) / Total Nitrogen
(mg/L) / NH4+-N
(mg/L) / Fermenting- bacterium
(ufc/mL)
JSA / Site J1 / 6.89±0.02 / 0.16±0.02 / 56.66±4.71 / 34.39±3.14 / 840±12
Site J2 / 7.81±0.01 / 0.19±0.02 / 46.77±4.44 / 27.55±2.35 / 1020±79
Site J3 / 7.3±0.02 / 0.14±0.03 / 29.95±2.12 / 15.35±1.21 / 540±17
JSB / Site J4 / 7.58±0.01 / 0.09±0.01 / 4.06±3.65 / 2.22±0.11 / NT
JSC / Site J5 / 7.91±0.02 / 0.12 ±0.01 / 15.41±1.27 / 7.24±0.42 / 520±14
Site J6 / 7.77±0.02 / 0.08±0.01 / 4.51±0.43 / 1.91±0.17 / 190±9
Site J7 / 7.94±0.01 / 0.11 ±0.01 / 4.37±0.34 / 1.18±0.18 / 420±24
Site J8 / 7.82±0.01 / 0.12±0.01 / 3.37±0.32 / 0.57±0.12 / 330±32

NT indicates that ferment bacteria were not detected at this site.

DNA Extraction and Detection of Antibiotic Resistance genes

20, 20, 20, 16, 9, 20 and 9 identified E. coli clones were selected from sites J1, J2, J3, J5, J6, J7 and J8, respectively and their DNA were extracted for the detection of antibiotic resistance genes and further analysis of fingerprint. The selected clones were cultured in LB liquid medium at 37 ºC for 8 h and then 200 μL cultures were collected and centrifuged at 5, 000g for 5 min. After washed with double distilled H2O (ddH2O) for twice, the pellets were re-suspended in 50 μL ddH2O. The suspended solution were heated at 95 ºC for 10 min and then centrifuged at 12,000g for 5 min. The supernate was used as templates for PCR amplification.

The PCR reaction mix (total 25 μL) contained 1μL template DNA, 2.5μL 10×PCR butter (Mg2+ free), 1.5μL Mg2+ (25mmol/L), 0.5μL dNTP (10mmol/L), 1U Taq polymerase, 2μL primer and ddH2O. PCR assays were carried out in a DNA Engine (Bio-Rad, USA) using the following conditions: a 5 min initial denature at 94℃ followed by 35 cycles of 1 min at 94℃, 45s at the annealing temperature (Table SI) and 1 min at 72℃, and a final extension step for 10 min at 72℃. Aliquots of PCR products of 4μL were mixed with 2μL loading butter dye and loaded on a horizontal agarose gel (1% (w/v) with Marker. DNA bands were stained with Gold view and then visualized by UV transillumination (UVP EC3, USA). To check reproducibility, PCR reactions were performed at least two times for each sample. The reaction was terminated with an extension step consisting of 7 min of incubation at 72 ºC. E. coli ATCC 25922 and all the reagents without any template were used as negative controls.

TABLE S2. PCRPrimers used in this study.

Resistant Type / primers / Target / Sequence (5`-3`) / Annealing temperature (°C) / Amplicon Size(bp) / Reference
Tetracycline / tet(A)-FW / tet(A) / GCTACATCCTGCTTGCCTTC / 60 / 210 / Ng et al., 2001
tet(A)-RV / CATAGATCGCCGTGAAGAGG
tet(B)-FW / tet(B) / TTGGTTAGGGGCAAGTTTTG / 58 / 659 / Ng et al., 2001
tet(B)-RV / GTAATGGGCCAATAACACCG
tet(C)-FW / tet(C) / CTTGAGAGCCTTCAACCCAG / 62 / 418 / Ng et al., 2001
tet(C)-RV / ATGGTCGTCATCTACCTGCC
tet(D)-FW / tet(D) / AAACCATTACGGCATTCTGC / 62 / 787 / Ng et al., 2001
tet(D)-RV / GACCGGATACACCATCCATC
tet(E)-FW / tet(E) / AAACCACATCCTCCATACGC / 60 / 278 / Ng et al., 2001
tet(E)-RV / AAATAGGCCACAACCGTCAG
tet(G)-FW / tet(G) / CAGCTTTCGGATTCTTACGG / 62 / 844 / Ng et al., 2001
tet(G)-RV / GATTGGTGAGGCTCGTTAGC
tet(K)-FW / tet(K) / TCGATAGGAACAGCAGTA / 54 / 169 / Ng et al., 2001
tet(K)-RV / CAGCAGATCCTACTCCTT
tet(L)-FW / tet(L) / TCGTTAGCGTGCTGTCATTC / 45 / 267 / Ng et al., 2001
tet(L)-RV / GTATCCCACCAATGTAGCCG
tet(M)-FW / tet(M) / GTGGACAAAGGTACAACGAG / 60 / 406 / Ng et al., 2001
tet(M)-RV / CGGTAAAGTTCGTCACACAC
tet(O)-FW / tet(O) / AACTTAGGCATTCTGGCTCAC / 45 / 515 / Ng et al., 2001
tet(O)-RV / TCCCACTGTTCCATATCGTCA
tet(S)-FW / tet(S) / CATAGACAAGCCGTTGACC / 58 / 667 / Ng et al., 2001
tet(S)-RV / ATGTTTTTGGAACGCCAGAG
tetA(P)-FW / tetA(P) / CTTGGATTGCGGAAGAAGAG / 58 / 676 / Ng et al., 2001
tetA(P)-RV / ATATGCCCATTTAACCACGC
tet(Q)-FW / tet(Q) / TTATACTTCCTCCGGCATCG / 60 / 904 / Ng et al., 2001
tet(Q)-RV / ATCGGTTCGAGAATGTCCAC
tet(X)-FW / tet(X) / CAATAATTGGTGGTGGACCC / 54 / 468 / Ng et al., 2001
tet(X)-RV / TTCTTACCTTGGACATCCCG
tet(30)-FW / tet(30) / CATCTTGGTCGAGGTGACTGG / 45 / 134 / Aminov et al., 2001
tet(30)-RV / ACGAGCACCCAGCCGAGC
tet(J)-FW / tet(J) / CGAAAACAGACTCGCCAATC / 55 / 184 / Aminov et al., 2001
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 / Aminov et al., 2002
tet(W)-RV / AGGTGTATCTATGATATTTAC
tet(Y)-FW / tet(Y) / ATTTGTACCGGCAGAGCAAAC / 45 / 181 / Aminov et al., 2002
tet(Y)-RV / GGCGCTGCCGCCATTATGC
tet(Z)-FW / tet(Z) / CCTTCTCGACCAGGTCGG / 55 / 204 / Aminov et al., 2002
tet(Z)-RV / ACCCACAGCGTGTCCGTC
Sulfonamide / sul(I)-FW / sul(I) / CGCACCGGAAACATCGCTGCAC / 45 / 163 / Pei et al., 2006
sul(I)-RV / TGAAGTTCCGCCGCAAGGCTCG
sul(II)-FW / sul(II) / TCCGGTGGAGGCCGGTATCTGG / 45 / 191 / Pei et al., 2006
sul(II)-RV / CGGGAATGCCATCTGCCTTGAG
sul(III)-FW / sul(III) / TCCGTTCAGCGAATTGGTGCAG / 45 / 128 / Pei et al., 2006
sul(III)-RV / TTCGTTCACGCCTTACACCAGC
sul(A)-FW / sul(A) / TCTTGAGCAAGCACTCCAGCAG / 45 / 299 / Pei et al., 2006
sul(A)-RV / TCCAGCCTTAGCAACCACATGG
β -lactamases / SHV-FW / SHV / GCGAAAGCCAGCTGTCGGGC / 45 / 304 / Henriques et al., 2006
SHV-RV / GATTGGCGGCGCTGTTATCGC
TEM-FW / TEM / AAAGATGCTGAAGATCA / 45 / 425 / Henriques et al., 2006
TEM-RV / TTTGGTATGGCTTCATTC
CTX-M-F / CTX / CGCTTTGCGATGTGCAG / 51 / 550 / Ahmed et al., 2007
CTX-M-R / ACCGCGATATCGTTGGT
CMY-F / CMY / GACAGCCTCTTTCTCCACA / 55 / 1015 / Ahmed et al., 2007
CMY-R / TGGAACGAAGGCTACGTA
OXA-F / OXA / TCAACTTTCAAGATCGCA / 45 / 591 / Ahmed et al., 2007
OXA-R / GTGTGTTTAGAATGGTGA
Oxy-F / Oxy / GGTTTTGGTAACTGTGACGGG / 45 / 725 / Ahmed et al., 2007
Oxy-R / CAGAGTGCAGAGTGTTGCAG
quinolone / qnrA-FW / qnrA / ATTTCTCACGCCAGGATTTG / 54 / 516 / Robicsek et al., 2006
qnrA-RV / GATCGGCAAAGGTTAGGTCA
qnrB-FW / qnrB / GATCGTGAAAGCCAGAAAGG / 56 / 469 / Robicsek et al., 2006
qnrB-RV / ACGATGCCTGGTAGTTGTCC
qnrS-FW / qnrS / ACGACATTCGTCAACTGCAA / 54 / 417 / Robicsek et al., 2006
qnrS-RV / TAAATTGGCACCCTGTAGGC
aac(6`)-Ib-F / aac(6`)-Ib / TTGCGATGCTCTATGAGTGGCTA / 50 / 482 / Robicsek et al., 2006
aac(6`)-Ib-R / CTCGAATGCCTGGCGTGTTT
Streptomycin / StrA-FW / StrA / TTGATGTGGTGTCCCGCAATGC / 57 / 383 / Hochhut et al., 2001
StrA -RV / CCAATCGCAGATAGAAGGCAA
StrB-FW / StrB / CCGCGATAGCTAGATCGCGTT / 48 / 470 / Ramachandran et al., 2007
StrS-RV / CGACTACCAGGCGACCGAAAT

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