Title Page
Contents
ABSTRACT 13
Chapter 1. Literature Review 16
1.1. Introduction 17
1.2. Symptoms of bovine mastitis 19
1.3. Types of bovine mastitis 20
1.4. Biofilm formation by bovine mastitis pathogens 21
1.5. Bovine mastitis pathogens 23
1.5.1. Staphylococcus aureus 25
1.5.2. Enterococcus faecalis 28
1.5.3. Pseudomonas aeruginosa 31
1.5.4. Escherichia coli 33
1.5.5. Evaluation of raw milk microbiota 36
1.6. Control of bovine mastitis: LAB-derived postbiotics 42
1.6.1. Organic acid 43
1.6.2. Bacteriocin 45
1.6.3. Hydrogen peroxide 48
1.7. Conclusion 50
Chapter 2. Prevalence and virulence characteristics of Enterococcus faecalis and Enterococcus faecium in bovine mastitis milk compared to normal raw milk 51
Abstract 52
2.1. Introduction 53
2.2. Materials and Methods 55
2.2.1. Experimental design 55
2.2.2. Sample collection and isolation of E. faecalis and E. faecium 57
2.2.3. DNA extraction and screening for virulence genes 57
2.2.4. Phenotype of virulence characteristics 60
2.2.5. Antibiotic resistance 61
2.2.6. Statistical analysis 62
2.3. Results 63
2.3.1. Identification of E. faecalis and E. faecium from BMM and NRM 63
2.3.2. Detection of virulence genes 65
2.3.3. Biofilm, gelatinase, and hemolysin production 67
2.3.4. Correlation between virulence genes and corresponding phenotype 69
2.3.5. Antibiotic resistance of E. faecalis and E. faecium 72
2.4. Discussion 74
2.5. Conclusion 78
Chapter 3. Screening of potential probiotics isolated from kefir and normal raw milk with anti-microbial activity against bovine mastitis pathogens 79
Abstract 80
3.1. Introduction 81
3.2. Materials and Methods 83
3.2.1. Experimental design 83
3.2.2. Isolation and identification of LAB 85
3.2.3. Survivability of LAB in simulated gastric and intestinal conditions 85
3.2.4. Virulence attributes of LAB 86
3.2.5. Physiological characteristics of LAB 86
3.2.6. Cytotoxicity of postbiotics treatment 88
3.2.7. Isolation and identification of bovine mastitis pathogens 89
3.2.8. Anti-microbial assay on bovine mastitis pathogens 90
3.2.9. Statistical analysis 90
3.3. Results 91
3.3.1. Screening LAB as potential probiotics 91
3.3.2. Assessment of physiological characteristics of LAB 96
3.3.3. Optimizing the concentration of postbiotics 98
3.3.4. Anti-microbial activity of postbiotics against bovine mastitis pathogens 100
3.4. Discussion 103
3.5. Conclusion 106
Chapter 4. Comparative anti-biofilm activities of postbiotics derived from kefir and normal raw milk lactic acid bacteria against bovine mastitis pathogens 107
Abstract 108
4.1. Introduction 109
4.2. Materials and Methods 111
4.2.1. Experimental design 111
4.2.2. Isolation and identification of LAB 113
4.2.3. Biochemical analysis 113
4.2.4. High-performance liquid chromatography (HPLC) analysis of postbiotics 113
4.2.5. Isolation and identification of bovine mastitis pathogens 114
4.2.6. Anti-biofilm assays on bovine mastitis pathogens 114
4.2.7. Mechanisms of anti-biofilm activities of postbiotic treatment 115
4.2.8. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 117
4.2.9. Statistical analysis 120
4.3. Results 121
4.3.1. Biochemical activities of isolated LAB 121
4.3.2. Organic acid-producing ability of isolated LAB 124
4.3.3. Anti-biofilm activities of postbiotics against bovine mastitis pathogens 126
4.3.4. Modulation of hydrophobicity, auto-aggregation, and EPS production of bovine mastitis pathogens 128
4.3.5. Modulation of biofilm-related genes in bovine mastitis pathogens 130
4.4. Discussion 133
4.5. Conclusion 138
References 139
Abstract (in Korean) 168
〈Table 1-1〉 Types of causative pathogens of bovine mastitis (4, 5). 24
〈Table 1-2〉 Prevalence and virulence characteristics of Staphylococcus aureus. 26
〈Table 1-3〉 Prevalence and virulence characteristics of Enterococcus faecalis. 29
〈Table 1-4〉 Prevalence and virulence characteristics of Pseudomonas aeruginosa. 32
〈Table 1-5〉 Prevalence and virulence characteristics of Escherichia coli. 34
〈Table 1-6〉 Case of organic acid in postbiotics as a novel anti-microbial agent for the prevention and treatment of bovine mastitis. 44
〈Table 1-7〉 Case of bacteriocins in postbiotics as a novel anti-microbial agent for the prevention and treatment of bovine mastitis. 46
〈Table 1-8〉 Case of hydrogen peroxide in postbiotics as a novel anti-microbial agent for the prevention and treatment of bovine mastitis. 49
〈Table 2-1〉 Polymerase chain reaction primers and product sizes for the detection of Enterococcus faecalis and Enterococcus faecium virulence genes. 59
〈Table 2-2〉 Distribution of virulence genes among Enterococcus faecalis isolates from bovine mastitis milk and normal raw milk. 66
〈Table 2-3〉 Correlation between virulence phenotype and genotype of Enterococcus faecalis and Enterococcus faecium isolated from bovine mastitis milk (BMM) and normal raw milk (NRM). 71
〈Table 2-4〉 Antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolates from bovine mastitis milk (BMM) and normal raw milk (NRM). 73
〈Table 3-1〉 Screening of 20 lactic acid bacteria strains as potential probiotics. 93
〈Table 3-2〉 Physiological characteristics of selected lactic acid bacteria. 97
〈Table 3-3〉 Identification of pathogens and somatic cell counts in bovine mastitis milk samples. 101
〈Table 4-1〉 Primer sequences used in gene expression analysis. 118
〈Table 4-2〉 Results of biochemical analysis of Lentilactobacillus kefiri LK1 and Enterococcus faecium EFM2 using the VITEK 2 122
〈Table 4-3〉 Organic acid concentrations in postbiotics. 125
〈Table 4-4〉 Hydrophobicity, auto-aggregation, and exopolysaccharide (EPS) production by bovine mastitis pathogens after treatment with postbiotics at 25% concentration. 129
〈Figure 1-1〉 Four processes of biofilm formation. 22
〈Figure 1-2〉 Alpha diversity indices of the microbiota in normal raw milk and bovine mastitis milk.... 39
〈Figure 1-3〉 Beta diversity of the microbiota in normal raw milk and bovine mastitis milk. (A) UPGMA tree (weighted unifrac), (B) weighted UniFrac principal coordinate analysis (PCoA), and (C) unweighted PCoA results.... 40
〈Figure 1-4〉 The relative taxonomy abundance ratios of normal raw milk and bovine mastitis milk. (A) phylum, (B) class, and (C) order. BMM : bovine mastitis milk; NRM : normal raw milk. 41
〈Figure 2-1〉 Flowchart of the experimental procedures. 56
〈Figure 2-2〉 Comparison of the prevalence of Enterococcus faecalis and Enterococcus faecium isolates in bovine mastitis milk (BMM; n=81) and normal raw milk (NRM; n... 64
〈Figure 2-3〉 Distribution of Enterococcus faecalis and Enterococcus faecium isolated from bovine mastitis milk (BMM) and normal raw milk (NRM) according to their virulence factors. (A) Biofilm formation ability and (B) gelatinase and hemolysin production.... 68
〈Figure 3-1〉 A schematic of the experimental design. LAB : Lactic acid bacteria; MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MALDI-TOF : Matrix-assisted laser... 84
〈Figure 3-2〉 Colorectal adenocarcinoma cell viability with postbiotics at 50%, 25%, 12.5%, and 6.25% concentrations. All data are expressed as mean ± standard deviation.... 99
〈Figure 3-3〉 Anti-microbial activities of postbiotics at 25% concentration against bovine mastitis pathogens. All data are expressed as mean ± standard deviation (n=5)... 102
〈Figure 4-1〉 Flow diagram of the experimental procedures. 112
〈Figure 4-2〉 Anti-biofilm activity of postbiotics at 25% concentration against (A) Staphylococcus aureus, (B) Enterococcus faecalis, (C) Pseudomonas aeruginosa, and... 127
〈Figure 4-3〉 Expression of genes related to hydrophobicity, auto-aggregation, and exopolysaccharide production of gram-positive bovine mastitis pathogens following postbiotic treatment at 25% concentration. (A-C) Staphylococcus aureus; (D-F) Enterococcus faecalis.... 131
〈Figure 4-4〉 Expression of genes related to hydrophobicity, auto-aggregation, and exopolysaccharide production of gram-positive bovine mastitis pathogens following postbiotic treatment at 25% concentration. (A-C) Pseudomonas aeruginosa; (D-F) Escherichia coli.... 132