김치에서 분리된 Leuconostoc mesenteroides 균주의 안전성 관련 유전체적 특성 분석

국문 초록 (Abstract)

발효식품에 적용 가능한 안전한 종균 후보를 선발하기 위해 김치에서 분리한 Leuconostoc mesenteroides 257개 균주를 대상으로 항생제 감수성 평가를 수행하였다. 항생제의 종류 및 cut-off value는 EFSA 가이드라인에 따라 설정하였으며, 그 결과 모든 균주는 ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin 및 tetracycline에 대해 감수성을 나타냈다. 반면, kanamycin과 streptomycin에 대해서는 각각 257개 균주(100%)와 249개 균주(96.9%)가 EFSA에서 제시한 cut-off value(kanamycin 16 mg/L, streptomycin 64 mg/L)를 초과하는 MIC 값을 보였다. 이에 kanamycin 및 streptomycin 내성이 Leu. mesenteroides의 내재형(intrinsic) 특성인지 여부를 확인하기 위해, 두 항생제에 대해 동시에 높은 MIC 값을 보인 균주 DMLM18과 낮은 MIC 값을 보인 대조 균주 DMLM17을 선별하여 전장유전체 분석을 수행하였다. 그 결과, kanamycin 내성과 관련된 것으로 알려진 aminoglycoside 6′-N-acetyltransferase (aacA) 유전자는 core 유전자로 확인된 반면, streptomycin 내성과 관련된 streptomycin 3″-adenylyltransferase (aadA) 유전자는 균주 특이적(accessory) 유전자로 확인되었다. 또한 aminoglycoside 계열 항생제 내성에 관여하는 것으로 보고된 core 유전자인 16S rRNA, rpsL, gidB 유전자 서열에서 내성과 연관된 변이가 추가적으로 확인되었다. 이러한 유전체 분석 결과를 EFSA의 항생제 감수성 평가 가이드라인에 근거하여 해석함으로써, 각 내성 관련 유전자가 내재형 인자인지 혹은 획득형 인자인지를 종합적으로 판단하였다.

To identify safe starter culture candidates for fermented foods, antimicrobial susceptibility was evaluated in 257 Leuconostoc mesenteroides strains isolated from kimchi. The antimicrobial agents tested and their corresponding cut-off values were determined in accordance with the European Food Safety Authority (EFSA) guidelines. As a result, all strains were susceptible to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, and tetracycline. In contrast, 257 strains (100%) and 249 strains (96.9%) exhibited minimum inhibitory concentrations (MICs) exceeding the EFSA cut-off values for kanamycin (16 mg/L) and streptomycin (64 mg/L), respectively. To determine whether resistance to kanamycin and streptomycin represents an intrinsic characteristic of Leu. mesenteroides, whole-genome sequencing was performed on strain DMLM18, which showed simultaneously high MICs for both antibiotics, and on strain DMLM17, which exhibited low MICs and served as a reference strain. Comparative genomic analysis revealed that the aminoglycoside 6′-N-acetyltransferase gene (aacA), which has been associated with kanamycin resistance, was identified as a core gene. In contrast, the streptomycin 3″-adenylyltransferase gene (aadA), known to contribute to streptomycin resistance, was identified as a strain-specific accessory gene. In addition, sequence variations associated with aminoglycoside resistance were identified in core genes encoding 16S rRNA, rpsL, and gidB. Based on these genomic findings and EFSA antimicrobial susceptibility assessment criteria, each resistance-associated determinant was further classified as either intrinsic or acquired.

다국어 초록 (Multilingual Abstract)

To identify safe starter culture candidates for fermented foods, antimicrobial susceptibility was evaluated in 257 Leuconostoc mesenteroides strains
isolated from kimchi. The antimicrobial agents tested and their corresponding cut-off values were determined in accordance with the European Food Safety Authority (EFSA) guidelines. As a result, all strains were susceptible to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, and tetracycline. In contrast, 257 strains (100%) and 249 strains (96.9%) exhibited minimum inhibitory concentrations (MICs) exceeding the EFSA cut-off values for kanamycin (16 mg/L) and streptomycin (64 mg/L), respectively. To determine whether resistance to kanamycin and streptomycin represents an intrinsic characteristic of Leu.
mesenteroides, whole-genome sequencing was performed on strain DMLM18, which showed simultaneously high MICs for both antibiotics, and on strain DMLM17, which exhibited low MICs and served as a reference strain. Comparative genomic analysis revealed that the aminoglycoside 6′-N-acetyltransferase gene (aacA), which has been associated with kanamycin resistance, was identified as a core gene. In contrast, the streptomycin 3″-adenylyltransferase gene (aadA), known to
contribute to streptomycin resistance, was identified as a strain-specific accessory gene. In addition, sequence variations associated with aminoglycoside resistance were identified in core genes encoding 16S rRNA, rpsL, and gidB. Based on these genomic findings and EFSA antimicrobial susceptibility assessment criteria, each resistance-associated determinant was further classified as either intrinsic or acquired.

목차 (Table of Contents)

• CONTENTS
• LIST OF TABLES ⅲ
• LIST OF FIGURES ⅳ
• 국문요약 ⅴ
• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 3
• 2.1 Bacterial strains and culture conditions 3
• 2.2 Antibiotic susceptibility 5
• 2.3 Genome sequencing 6
• 2.4 Comparative genome analysis 7
• 2.5 Nucleotide sequence accession number 7
• Ⅲ. Results 8
• 3.1 Antibiotic resistance profiles of Leuconostoc mesenteroides 8
• 3.2 Genome properties of Leuconostoc mesenteroides
• strain DMLM17 and DMLM18 11
• 3.3 Genome insights for kanamycin and streptomycin resistance
• of strain DMLM17 and DMLM18 13
• 3.3.1 aacA gene and kanamycin resistance as an intrinsic trait 13
• 3.3.2 aadA gene and streptomycin resistance as an acquired trait 16
• 3.4 Effect of other factors for kanamycin and streptomycin resistance 19
• Ⅳ. Conclusion 30
• References 32
• ABSTRACT 39