Antibiotic Growth Promoter Olaquindox Increases Pathogen Susceptibility in Fish by Inducing

Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis

Suxu He, Quanmin Wang, Shuning Li, Chao Ran, Xiaoze Guo, Zhen Zhang, Zhigang Zhou*

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China;

* Tel.: +86-10-82106073; Fax: +86-10-82106054; E-mail:

Supplemental files

Methods

Total gut bacterial count of zebrafish digesta.

After the satiation feeding, the gut digesta were sampled at 0, 2, 4, 6 and 8 h. The total DNA were extracted and primers GB (general bacteria) 1114f and 1275r (Supplemental Table 2) were employed to quantify the total bacteria counts by real-time PCR.

Culturing and characterization of OLA discriminatory bacterial strains.

After the OLA pretreated for 14 days, the gut digesta microbial from donor fish were harvested from euthanized fish by rapidly removing the gut and then manually extruding the contents into a vial containing sterile argonsparged PBS. Gut content samples were resuspended in lysogeny broth (aerobic) and brain-heart infusion-blood (anaerobic) agar for 24 h at 28°C. Isolated colonies were picked into single wells of a deep 96-well plate, with 600µl liquid culture per well, and grown anaerobically or aerobicly at 28°C overnight. Taxonomies were confirmed by sequencing full-length 16S rRNA amplicons produced using primers 27F and 1391R.

Transfer of marker taxa to GF zebrafish.

The marker species were chosen based on the pure culture sequence results and metagenomics sequence result, the main culture strains were firstly picked out rely on the 16S rRNA sequence, then check these strains whether as the main changed strains on genus level from the metagenomics result, 6 strains were chosen for transfer to GF zebrafish (Table S3). 400 gnotobiotic larvae at72 hpf were immersed in a final concentration of bacterial strain (Table S3) at 106 CFU/mL. At 6 dpf, the treated larvae were detected ROS activity as described in the main text.

Table S1 Feed formulation and chemical composition

Composition / Percent（%）
Casein / 40.00
Gelatin / 10.00
Dextrin / 35.00
Soybean oil / 6.00
Vc phosphate ester / 0.10
Vitamin mix1 / 0.20
Mineral mix2 / 0.20
Ca(H2PO4)2 / 2.00
Choline chloride / 0.20
Sodium alginate / 2.00
Microcrystalline cellulose / 3.97.
Lysine / 0.33
Chemical composition
Crude protein / 42.19
Crude lipid / 6.09
Ash / 8.11
Gross energy (Kj g-1 DM) / 18.55

1Vitamin mix, given as g kg−1: vitamin A acetate (500000 IU g−1) 1.0, vitamin D3 (500000 IU g−1) 2.5, dl-α-tocopheryl acetate (50%) 20.0, vitamin K3 sodium bisulphite (50%) 1.0, vitamin C phosphate (25%) 60.0, choline-hydrogtatrat (100%) 400.0, thiamine nitrate (100%) 1.5, riboflavin (80%) 1.88, pyridoxine hydrochloride (100%) 2.0, nicotinic acid (100%) 20.0, inositol (100%) 40.0, folic acid (100%) 0.5, Ca-pantothenate (100%) 6.0, biotin (2%) 7.5.

2Mineral mix given as g kg−1: MgSO4*7H2O 3.0, O4SZn*7H2O 0.15, FeSO4*7H2O 0.5, MnSO4*H2O 0.35, CuSO4*5H2O 0.03, KH2PO4 20.7, CaCO3 14.8, NaCl 6.0, KCl 1.0, KJO3 0.01, Na2MoO4*2H2O 0.0083, CoCl2*6H2O 0.0017, Na2SeO3*H2O 0.0002.

Table S2 Primers for real-time PCR

Gene / Sequence / Accession NO.
R / CGG CAG GCT GTA GTA TTG CTC
Il1β / F / CATCAAACCCCAATCCACAG / NM_212844.2
R / CACCACGTTCACTTCACGCT
Il10 / F / ACGAGATCCTGCATTTCTACTTG / AY887900.1
R / AGCTCCCCCATAGCTTTATAGAC
iNos / F / CCA GAG CCT TCG TCT GGA GA / KJ679875.1
R / TTA GAG CCT GGA CGA GCG TG
Ndi / F / TACAGAGGGGGAATCAGAAC / AC024175.3
R / TTGGTCGTATCGGAATCGT
Mpo / F / TCCAAAGCTATGTGGGATGTGA / NM_212779.1
R / GTCGTCCGGCAAAACTGAA
Il8 / F / GTCGCTGCATTGAAACAGAA / XM_001342570.6
R / CTTAACCCATGGAGCAGAGG
Cclc25ab / F / AGCACCTCTCGCTTTGTGTT / NM_001115056.1
R / TGTTTGAAAGGCACTTGACG
Hsp70 / F / CTGGACTGAATGTTGCTCGC / NM_001113589.1
R / CAGATGAGTGTCTCCAGCGG
Saa / F / CGCAGAGGCAATTCAGAT / NM_001005599.1
R / CAGGCCTTTAAGTCTGTATTTGTTG
Cfb / F / GCCACAGTGCTACGCTGATTT / NM_131338.2
R / GTTGAACTGTTAGAGTTGTCGTTAGAGAATT
Rps11 / F / ACAGAAATGCCCCTTCACTG / NM_213377.1
R / GCCTCTTCTCAAAACGGTTG
16S rRNA gene 1114F and 1275R / F / CGGCAACGAGCGCAACCC
R / CCATTGTAGCACGTGTGTAGCC

Figure S1 The total number of bacteria in gut digesta of zebrafish after satiation feeding. Gut digesta were sampled at 0, 2, 4, 6 and 8 h post feeding. n=5 fish sample at each time point. An asterisk indicates a significant difference (** P<0.001) between the 0 h and 6 h sample.

Figure S2 Quantitative PCR analysis of the expression of cytokine genes in GF larvae colonized

by different microbiotas at 106 CFU/mL. Each bar represents the average from eight individual

larvae. Data are representative of 3 independent experiments. An asterisk indicates a significant

difference (*at P<0.05,** P<0.001) between the control and antibiotic-treated group isolated

strains. cfb, complement factor b; Cclc25ab, CC motif chemokine C25ab; IL8, Interleukin-8 ;

Saa,serum amyloid a; MPO, myeloperoxidase.

Figure S3 Microbial composition changes in the gut digesta of zebrafish fed OLA-supplemented diet for fourteen days. (A) Relative abundance of the phyla in the gut digesta; (B) Heat map showing the relative abundance of main bacteria identified at the family level.

At 6 h post the final feeding, 24 fish were randomly selected from each group. To avoid individual differences, gut digesta samples from 12 fish were pooled together.