Özcan Ali, Kiraz Deniz, Yeniyiğit Çağlar, Gül İlker
Animal Originated Foodstuffs Department, Central Research Institute of Food and Feed Control, Bursa, Turkey.
Curr Microbiol. 2025 Aug 20;82(10):467. doi: 10.1007/s00284-025-04373-0.
In this study, we developed a novel 13 housekeeping gene multilocus sequence typing (MLST) scheme for Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) using whole-genome sequencing data, comprising 218 GenBank-retrieved and 29 newly isolated Turkish strains. We identified 291 distinct alleles across 13 housekeeping genes (clpX, pepX, pheS, mutL, murE, dnaA, gyrB, ddlA, pyrG, rpoB, fusA, recA, and tuf), with allele frequencies ranging from 2.7 to 10.9%, and defined 133 sequence types (STs), including 12 novel STs (ST122-ST133) unique to Turkish isolates, demonstrating superior discriminatory power compared to prior 8-gene MLST schemes (106 STs). This scheme offers a practical, standardized, and less computationally demanding alternative to core genome MLST (cgMLST) for large-scale epidemiological and industrial tracking, while maintaining high resolution. Population structure analysis revealed 17 clonal complexes (CC1-CC17) and 76 singleton STs (57.1%), with the largest complex (CC-1) containing 17 STs. Turkish isolates formed a distinct clonal complex (CC-6) or existed as singletons, underscoring their genetic uniqueness. Genetic diversity analyses showed significant differences between reference and Turkish populations, with nucleotide diversity (π) values of 0.00364 and 0.00225, respectively. The gyrB locus exhibited the highest diversity in reference strains (π = 0.00858), while pyrG and mutL showed the highest diversity in Turkish isolates. Significant linkage disequilibrium was detected in both populations ( = 0.2653 for reference strains; = 0.3395 for Turkish isolates), suggesting predominantly clonal population structures with limited recombination. Split decomposition and Phi test analyses confirmed the presence of recombination (P < 0.05) across the concatenated sequences. These findings enhance our understanding of L. bulgaricus population genetics and highlight the importance of geographical diversity in shaping bacterial evolution. This standardized MLST scheme provides a robust, scalable, and portable framework for starter culture selection and the tracking of traditional dairy microbiota, complementing higher-resolution whole-genome typing methods.
在本研究中，我们利用全基因组测序数据，为德氏乳杆菌保加利亚亚种（保加利亚乳杆菌）开发了一种新型的13个管家基因多位点序列分型（MLST）方案，该方案涵盖了从GenBank获取的218株菌株以及29株新分离的土耳其菌株。我们在13个管家基因（clpX、pepX、pheS、mutL、murE、dnaA、gyrB、ddlA、pyrG、rpoB、fusA、recA和tuf）中鉴定出291个不同的等位基因，等位基因频率范围为2.7%至10.9%，并定义了133种序列类型（STs），其中包括12种土耳其分离株特有的新型STs（ST122 - ST133），与之前的8基因MLST方案（106种STs）相比，显示出更强的鉴别能力。该方案为大规模流行病学和工业追踪提供了一种实用、标准化且计算要求较低的替代核心基因组MLST（cgMLST）的方法，同时保持了高分辨率。群体结构分析揭示了17个克隆复合体（CC1 - CC17）和76个单例STs（57.1%），其中最大的复合体（CC - 1）包含17种STs。土耳其分离株形成了一个独特的克隆复合体（CC - 6）或作为单例存在，突出了它们的遗传独特性。遗传多样性分析表明，参考群体和土耳其群体之间存在显著差异，核苷酸多样性（π）值分别为0.00364和0.00225。gyrB基因座在参考菌株中表现出最高的多样性（π = 0.00858），而pyrG和mutL在土耳其分离株中表现出最高的多样性。在两个群体中均检测到显著的连锁不平衡（参考菌株的 = 0.2653；土耳其分离株的 = 0.3395），表明主要是克隆群体结构，重组有限。分裂分解和Phi检验分析证实了在串联序列中存在重组（P < 0.05）。这些发现增进了我们对保加利亚乳杆菌群体遗传学的理解，并突出了地理多样性在塑造细菌进化中的重要性。这种标准化的MLST方案为发酵剂培养物选择和传统乳制品微生物群追踪提供了一个强大、可扩展且便携的框架，补充了更高分辨率的全基因组分型方法。
