Supporting information

Combination of antibiotics suppressed the increase of a part of ARGs in fecal microorganism of weaned pigs

Huizhi Li, Qingpo Chu, Feilong Xu, Lingling Fu, Tingting Liang, Yuan Li, and Bo Zhou*

College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

*Corresponding author at: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. Tel.: +86 25 84395362.

E-mail:

Details of Sample Collection

All feces samples were collected in the Yong-kang Agricultural and Animal Husbandry Co., Ltd. in Jiangsu province, China (31.74ºN) were collected in October 2014. Fresh feces were collected within minutes after defecation and kept sterile to avoid cross-contamination. The feces samples were immediately Stored in the refrigerator to 20 °C in the Farm. All feces samples were stored in darkness and maintained with ice packets returned to the laboratory , where they were stored at -20 °C before DNA extraction (within 24 hours).

Table S1 Equations of standard curves for qPCR

Gene name / Equations / R2
16S rRNA / y=-0.26282x+18.80749 / 0.9983
tetA / y=-0.25212x+18.53780 / 0.9986
tetC / y=-0.27261x+18.57631 / 0.9994
tetL / y=-0.22048x+17.90437 / 0.9992
tetO / y=-0.27696x+18.74736 / 0.9999
tetW / y=-0.29623x+18.70442 / 0.9998
tetX / y=-0.27883x+18.78745 / 0.9998

Table S2: PCR detection of selected tet genes in feces samples

Sample* / tetA / tetC / tetD / tetG / tetL / tetM / tetO / tetQ / tetW / tetX
CT1 / + / + / + / - / + / + / + / + / + / +
CT2 / + / + / + / - / + / + / + / - / + / +
CT3 / + / + / + / - / + / + / - / + / + / -
CT4 / + / + / + / - / + / + / - / - / + / -
CT5 / + / + / + / - / + / + / - / - / + / -
CT6 / + / + / + / - / + / + / - / - / + / +
CT7 / + / + / + / + / + / + / - / - / + / +
CT8 / + / + / + / + / + / + / - / - / + / +
CT9 / + / + / + / + / + / + / - / - / + / +
CBC1 / + / + / + / - / + / + / + / - / + / +
CBC2 / + / + / + / - / + / + / + / - / + / -
CBC3 / + / + / + / - / + / + / + / - / + / +
CBC4 / + / + / + / - / + / + / + / - / + / +
CBC5 / + / + / + / - / + / + / + / + / + / +
CBC6 / + / + / + / + / + / + / + / - / + / +
CBC7 / + / + / + / + / + / + / + / + / + / +
CBC8 / + / + / + / - / + / + / + / + / + / +
CBC9 / + / + / + / - / + / + / + / - / + / -
BC1 / + / + / + / - / + / + / + / - / + / -
BC2 / - / + / - / - / + / + / + / - / + / -
BC3 / + / + / - / - / + / + / + / - / + / -
BC4 / + / + / - / + / + / + / + / + / + / +
BC5 / + / + / + / + / + / - / + / + / + / -
BC6 / + / + / + / + / + / + / + / + / + / +
BC7 / + / + / + / - / + / - / - / - / + / -
BC8 / + / + / + / + / + / + / + / + / + / +
BC9 / + / + / + / + / + / + / + / + / + / +
CTC1 / + / + / + / + / + / + / + / + / + / +
CTC2 / + / + / - / - / + / + / + / - / + / +
CTC3 / + / + / + / - / + / + / + / - / + / +
CTC4 / + / + / + / - / + / + / + / - / + / +
CTC5 / + / + / + / - / + / + / + / - / + / -
CTC6 / + / + / + / - / + / + / + / - / + / -
CTC7 / + / + / - / + / + / + / + / + / + / +
CTC8 / + / + / + / - / + / + / + / + / + / +
CTC9 / + / + / + / + / + / + / + / + / + / +
Frequency / 35/36 / 36/36 / 31/36 / 13/36 / 36/36 / 34/36 / 28/36 / 14/36 / 36/36 / 24/36
“+”: positive; “-“: negative.

*: The samples were designated as below: CT1-CT9, the feces sample form CT group (basal diet, no antibiotics); CBC1-CBC9, the feces sample form CBC group (supplement chlortetracycline, bacitracin zinc and colistin sulphate in basal diet); BC1-BC9, the feces sample form BC group (supplement bacitracin zinc and colistin sulphate in basal diet); CTC1-CTC9, the feces sample form CTC group (supplement chlortetracycline in basal diet).

Table S3: qPCR detection of selected tet genes in feces samples

Gene name / CT / CBC / BC / CTC
tetA/16S rRNA / (1.59±0.15)×10-4 / (4.40±0.98)×10-4 / (3.49±0.38)×10-4 / (2.72±0.49)×10-4
tetC/16S rRNA / (1.93±0.56) ×10-3 / (2.09±0.51)×10-3 / (1.33±0.33)×10-3 / (6.28±0.15)×10-3
tetL/16S rRNA / (8.09±1.44)×10-4 / (1.34±0.22)×10-3 / (1.65±0.28)×10-3 / (5.40±0.95)×10-3
tetO/16S rRNA / (6.62±0.20)×10-2 / (1.08±0.24)×10-1 / (1.24±0.67)×10-1 / (1.05±0.84)×10-2
tetW/16S rRNA / (3.60±0.24)×10-2 / (4.30±0.47)×10-2 / (3.48±0.24)×10-2 / (5.73±0.43)×10-2
tetX/16S rRNA / (9.51±0.84)×10-2 / (1.03±0.10)×10-1 / (1.78±0.27)×10-1 / (2.00±0.40)×10-1

CT: basal diet (no antibiotics); CBC: 75mg/kg Chlortetracycline, 40mg/kg Bacitracin Zinc, 20mg/kg Colistin Sulphate and basal diets; BC: 40mg/kg Bacitracin Zinc, 20mg/kg Colistin Sulphate and basal diets; CTC: 75mg/kg Chlortetracycline and basal diets.The datas are presented as mean ± SEM

Table S4: The Ct values of Negative control and ten-fold serial dilutions of the plasmids (Standards) quantified by qPCR

Dilutions / 16S / tetA / tetC / tetL / tetO / tetW / tetX
Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct / Standards
(ng/μl) / Ct
Initial / 2.0×101 / 8.05 / 2.04×101 / 7.68 / 1.88×101 / 7.04 / 2.0×101 / 5.36 / 2.14×101 / 7.31 / 2.0×101 / 6.89 / 2.0×101 / 7.59
101-fold / 2.0×100 / 11.79 / 2.04×100 / 11.30 / 1.88×100 / 10.46 / 2.0×100 / 9.68 / 2.14×100 / 10.85 / 2.0×100 / 10.03 / 2.0×100 / 10.78
102-fold / 2.0×10-1 / 15.24 / 2.04×10-1 / 14.66 / 1.88×10-1 / 13.89 / 2.0×10-1 / 14.37 / 2.14×10-1 / 14.45 / 2.0×10-1 / 13.29 / 2.0×10-1 / 14.47
103-fold / 2.0×10-2 / 18.98 / 2.04×10-2 / 18.70 / 1.88×10-2 / 17.79 / 2.0×10-2 / 18.75 / 2.14×10-2 / 17.97 / 2.0×10-2 / 16.73 / 2.0×10-2 / 18.08
104-fold / 2.0×10-3 / 23.27 / 2.04×10-3 / 22.69 / 1.88×10-3 / 21.37 / 2.0×10-3 / 23.54 / 2.14×10-3 / 21.47 / 2.0×10-3 / 20.27 / 2.0×10-3 / 21.75
105-fold / 2.0×10-4 / 27.63 / 2.04×10-4 / 27.06 / 1.88×10-4 / 25.08 / 2.0×10-4 / 28.49 / 2.14×10-4 / 25.35 / 2.0×10-4 / 23.64 / 2.0×10-4 / 25.32
106-fold / 2.0×10-5 / 31.02 / 2.04×10-5 / 30.90 / 1.88×10-5 / 28.56 / 2.0×10-5 / 32.78 / 2.14×10-5 / 28.92 / 2.0×10-5 / 26.99 / 2.0×10-5 / 28.72
NC* / 0.0 / 36.69 / 0.0 / 36.50 / 0.0 / 37.04 / 0.0 / 36.23 / 0.0 / 37.01 / 0.0 / 36.44 / 0.0 / 36.53

*NC: Negative control