Department of Medicine and Health Sciences "Vincenzo Tiberio", University of Molise, Italy.
Int J Food Microbiol. 2019 Nov 16;310:108289. doi: 10.1016/j.ijfoodmicro.2019.108289. Epub 2019 Aug 6.
This study aimed to characterize serovar 1/2a, 1/2b, 1/2c and 4b of Listeria monocytogenes cultures based on High Resolution Melting (HRM) profiles, targeting 53 fragments in the region comprising prs, Listeria Pathogenicity Island-1 (LIPI-1) and ldh loci, and 28 fragments of inlAB operon. Fifty L. monocytogenes cultures (28 of lineage I, 22 of lineage II) were tested. Real time PCR and EvaGreen-based HRM assays were performed, and the HRM profile for each amplicon was compared to that of L. monocytogenes EGD-e strain. Considering all fragments tested, 45 HRM profiles were identified (Diversity Index = 99.35). Similarity analysis identified two main clusters: the first consisted of 25 cultures, including all 1/2a and 1/2c strains, except for three isolates from food of serovar 4b; the second group only included 1/2b and 4b isolates. Eighteen out of targeted amplicons showed constant HRM characteristics irrespective of the serovar/lineage, and hlyA displayed the most stable melting behavior. Conversely, thirteen amplicons were specific for 1/2b and 4b isolates, showing major variations within actA, followed by prs, prfA, mpl and plcB genes. A fragment targeting an intragenic region (part of plcA and part of an unknown gene) had a melting profile allowing differentiation between 4b and 1/2b isolates showing different Tm variants. Amplicons of inlA and inlB exhibited the largest intra-strain melting differences, and one inlB fragment could allow discriminating between 4b and 1/2b cultures, as well as between lineages. A greater level of genetic diversity amongst 1/2a cultures compared to 1/2c, 1/2b and 4b isolates was observed, with variations identified in LIPI-1, as well as within inlA and inlB genes. Sequencing analysis of amplicons with differential HRM profile from EGD-e confirmed point mutations. The study findings underlines that HRM-based approach may be useful for bacterial subtyping for epidemiological purposes when sequencing-based methods are not available.
本研究旨在基于高分辨率熔解（HRM）图谱对血清型 1/2a、1/2b、1/2c 和 4b 的李斯特菌进行特征描述，该图谱针对包含 prs、李斯特菌致病性岛-1（LIPI-1）和 ldh 基因座的 53 个片段以及 inlAB 操纵子的 28 个片段进行靶向分析。共检测了 50 株李斯特菌培养物（谱系 I 28 株，谱系 II 22 株）。进行了实时 PCR 和 EvaGreen 基 HRM 检测，并将每个扩增子的 HRM 图谱与李斯特菌 EGD-e 株的图谱进行比较。考虑到所有测试的片段，共鉴定出 45 种 HRM 图谱（多样性指数为 99.35）。相似性分析确定了两个主要聚类：第一个聚类包含 25 株培养物，包括所有 1/2a 和 1/2c 菌株，但 4b 血清型的 3 株分离株除外；第二个聚类仅包含 1/2b 和 4b 分离株。18 个目标扩增子的 HRM 特征在血清型/谱系之间保持不变，hlyA 显示出最稳定的熔化行为。相反，13 个扩增子是 1/2b 和 4b 分离株的特异性扩增子，在 actA 中显示出主要的变异，其次是 prs、prfA、mpl 和 plcB 基因。针对内含子区域（plcA 的一部分和一个未知基因的一部分）的靶片段具有允许区分具有不同 Tm 变体的 4b 和 1/2b 分离株的熔化图谱。inlA 和 inlB 扩增子的菌株内熔化差异最大，一个 inlB 片段可以区分 4b 和 1/2b 培养物，以及谱系。与 1/2c、1/2b 和 4b 分离株相比，1/2a 培养物的遗传多样性水平更高，在 LIPI-1 以及 inlA 和 inlB 基因中都发现了变异。对 EGD-e 中具有差异 HRM 图谱的扩增子进行测序分析证实了点突变。研究结果表明，当无法使用测序方法时，基于 HRM 的方法可能有助于流行病学目的的细菌亚型分型。
