Title Page
Contents
Abstract 11
1. Introduction 13
2. Materials and methods 17
2.1. Experiment design 17
2.2. Microbial community analysis 18
2.2.1. Culture-dependent analysis of fecal bacteria 18
2.2.2. Culture-independent analysis using the Illumina iSeq system 21
2.3. Quantification of antibiotic resistance genes 23
2.4. Data analysis 27
2.4.1. Bioinformatic analysis 27
2.4.2. Correlation analysis 29
2.4.3. Statistical analysis 30
3. Results 31
3.1. Fish performance 31
3.2. Dynamics in fecal microbiome 33
3.2.1. Enumeration and composition of cultured bacteria 33
3.2.2. Microbiome diversity through Illumina iSeq sequencing 42
3.2.3. Microbiome community based on Illumina iSeq sequencing 49
3.3. Dynamics in ARGs and intI1 58
3.4. Correlation of ARGs and intI1 68
3.5. Relationship between ARGs, intI1, and microbial community 70
4. Discussion 79
국문요약 89
References 91
Table 1. The list of primer sets used in qPCR array. 25
Table 2. Mean±standard deviation length and weight measurements for control, oxytetracycline (OTC)-, sulfadiazine/trimethoprim... 32
Table 3. Bacterial species isolated from rainbow trout feces during the experiment period. The cultured bacteria were identified by comparing them with the closest type strains using the RDP SeqMatch program. 36
Table 4. Mean±standard deviation abundance (%) of cultured bacterial phyla in the feces of rainbow trout from the control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated groups before, during, and after... 40
Table 5. Good's coverage of rainbow trout fecal samples. Each value represents the mean±standard deviation of good's coverage. 44
Table 6. Mean±standard deviation abundance (%) of the 10 most abundant bacterial phyla in the feces of rainbow trout from the control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated groups. 53
Table 7. Mean±standard deviation abundance (%) of the 25 most abundant bacterial genera in the feces of rainbow trout from the control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated groups. 54
Table 8. Mean±standard deviation relative abundance antibiotic resistance genes and intl1 in the feces of rainbow trout from the control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated groups. 62
Table 9. Pearson correlation levels between intl1 and antibiotic resistance genes in the feces of control, oxytetracycline (OTC)-, and... 69
Table 10. Presence of antibiotic resistance genes (ARGs) in genera. The table shows the harbored (+) or not harbored (-) of ARGs in differently abundant genera in the fecal microbiota of rainbow trout between control and... 77
Figure 1. Schematic plan illustrating the treatment and withdrawal period. Rainbow trout were fed diets supplemented with oxytetracycline (OTC) or sulfadiazine/trimethoprim (SDZ/TMP) for 10 consecutive days.... 19
Figure 2. The changes in the counts of cultured bacteria in the feces of control rainbow trout, as well as those treated with oxytetracycline (OTC),... 34
Figure 3. Relative abundance (%) of cultured bacterial phyla in the feces of rainbow trout from the control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated groups before, during, and after antibiotic... 39
Figure 4. Rarefaction curves showing observed OTUs richness in samples. The x-axis represents the sequencing depth in the number of reads obtained from control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-... 43
Figure 5. Alpha diversity (Observed OTUs, Chao1, Inverse Simpson, Shannon index) measures of microbiome communities in the feces of... 46
Figure 6. Beta diversity according to treatment-by-time (A, B), treatment (C), and time (D) in the feces of control, oxytetracycline (OTC)-, and... 48
Figure 7. Relative abundance of the 10 most abundant bacterial phyla (A) and 25 most abundant bacterial genera (B) in the feces of control, oxytetracycline (OTC)-, and sulfadiazine/trimethoprim (SDZ/TMP)-treated rainbow trout. 52
Figure 8. Linear discriminant analysis effect size (LEfSe) analysis identifying taxonomic differences in the fecal microbiota at phylum and... 57
Figure 9. Relative abundance of fifteen antibiotic resistance genes and intl1 in rainbow trout feces before, during, and after oxytetracycline... 61
Figure 10. Association network among antibiotic resistance genes (ARGs), mobile genetic element (MGE), and fecal microbiomes. Genus correlated with tetracycline, sulfonamide, trimethoprim resistance genes, and intl1 (A); with... 75