Biochemical and Molecular Biological Elucidation of Antibiotics-Resistance Mechanism of Probiotic Strains = 프로바이오틱스 균주의 항생제 내성 기작에 대한 생화학적·분자생물학적 규명

다국어 초록 (Multilingual Abstract)

Understanding antibiotic resistance in probiotic lactic acid bacteria (LAB) is essential for ensuring their safety in food, fermentation, and industrial applications. In this study, a comprehensive investigation was conducted to elucidate the resistance mechanisms of Pediococcus acidilactici SY21 and Lentilactobacillus buchneri KU200793, two LAB strains previously identified as exhibiting resistance to multiple antibiotics. A wide range of physiological, biochemical, and molecular techniques was employed to systematically evaluate canonical and noncanonical resistance pathways. Minimum inhibitory concentration (MIC) assays were first performed to confirm phenotypic resistance profiles relative to EFSA microbiological cut-off values. To determine whether enzymatic antibiotic inactivation contributed to resistance, HPLC- based time-course analyses were conducted using both culture supernatants and cell lysate fractions. Ribosomal proteins and rRNA sequences were compared with those of susceptible reference strains to investigate the presence of target-site modifications. Cell envelope permeability was assessed through alkaline phosphatase activity, extracellular protein leakage, and potassium efflux, providing insight into potential structural or membrane-associated mechanisms. The involvement of efflux pumps was evaluated through assays using multiple chemical inhibitors, while transcriptomic and qPCR analyses were performed for P. acidilactici SY21 to characterize antibiotic- specific gene expression responses associated with translational stress. Through these integrated approaches, distinct resistance strategies were identified. P. acidilactici SY21 exhibited kanamycin and clindamycin resistance mainly through physiological stress-response pathways, including the induction of molecular chaperones and ABC-F–mediated ribosomal protection, rather than through target-site alterations, efflux activation, or clear enzymatic inactivation. KU200793, in contrast, displayed strong tetracycline resistance primarily driven by efflux pump activation, as demonstrated by pronounced MIC reductions in the presence of inhibitors. Both strains lacked ribosomal mutations or cell envelope disruption, and HPLC analyses indicated that enzymatic degradation, although possible, was not a major contributor. Collectively, this study revealed that probiotic LAB may utilize noncanonical adaptive resistance mechanisms that differ from those described in pathogenic bacteria. These findings highlight the need to incorporate mechanism-based evaluations—including stress-response pathways and efflux activity—into safety assessment frameworks for probiotic strains used in food and industrial applications.

목차 (Table of Contents)

• 1. Introduction 13
• 1.1. Probiotics 13
• 1.2. Antibiotics 15
• 1.3. Mechanisms of antibiotic resistance 17
• 1.4. Antibiotic-resistant probiotic strains 20
• 1.5. Aim of this study 23
• 2. Materials and methods 24
• 2.1. Bacterial culture and antibiotic treatment 24
• 2.2. Antibiotic resistance evaluation by broth microdilution assay 26
• 2.3. Enzymatic inactivation of antibiotics 27
• 2.3.1. Kanamycin inactivation assay using HPLC 27
• 2.3.2. Clindamycin inactivation assay using HPLC 29
• 2.3.3. Tetracycline inactivation assay using HPLC 31
• 2.4. Sequence-based evaluation of ribosomal target modifications 33
• 2.4.1. Kanamycin: comparison of 30S ribosomal protein S12 (RpsL) sequences 34
• 2.4.2. Clindamycin: sequence analysis of ribosomal proteins L3 (RplC), L4 (RplD), and L22 (RplV) 35
• 2.4.3. Tetracycline: comparison of 30S ribosomal protein S10 (RpsJ) sequences 36
• 2.5. Sequence-based identification of rRNA mutations associated with antibiotic resistance 37
• 2.5.1. Kanamycin: analysis of 16S rRNA mutations 38
• 2.5.2. Clindamycin: analysis of 23S rRNA domain V mutations 39
• 2.5.3. Tetracycline: analysis of 16S rRNA mutations 40
• 2.6. Cell wall permeability 41
• 2.7. Cell membrane permeability 42
• 2.7.1. Extracellular protein assay 42
• 2.7.2. Extracellular K⁺ determination 43
• 2.8. Efflux pump activation test 44
• 2.9. Transcriptomic and quantitative gene expression analysis 45
• 2.9.1. Transcriptomic analysis (RNA-seq) 45
• 2.9.2. Total RNA extraction and complementary DNA (cDNA) synthesis 46
• 2.9.3. Real Time–quantitative PCR (real time-qPCR) 48
• 3. Results 52
• 3.1. Determination of MICs of probiotic strains 52
• 3.2. HPLC-based assessment of antibiotic degradation in probiotic strains 54
• 3.3. Ribosomal target modification analysis 56
• 3.4. Sequence-based analysis of rRNA mutations associated with antibiotic resistance 60
• 3.5. Cell membrane permeability 65
• 3.5.1. Extracellular protein assay 65
• 3.5.2. Extracellular K⁺ determination 67
• 3.6. Alkaline phosphatase (AKP) activity 69
• 3.7. Efflux pump activation test 71
• 3.8. RNA-seq analysis 77
• 3.9. Quantitative analysis of mRNA expression in P. acidilactici SY21 under antibiotic stress 80
• 4. Discussion 85
• 4.1. Pediococcus acidilactici SY21 85
• 4.2. Lentilactobacillus buchneri KU200793 97
• 5. Conclusion 101
• References 105
• 국문 초록 114