1株三文鱼源ST18型单核细胞增生李斯特氏菌LMSUR02的耐药性、血清型及全基因组测序分析
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1.中国海洋大学食品科学与工程学院;2.中国水产科学研究院黄海水产研究所, 农业农村部水产品质量安全检测与评价重点实验室

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TS254.7

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中央级公益性科研院所基本科研业务费专项基金


Antibiotic Resistance, Serotype and Whole Genome Sequencing Analysis of a ST18 Listeria monocytogenes LMSUR02 Isolated from Salmon
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1.College of Food Science and Engineering, Ocean University of China;2.Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences

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    摘要:

    单核细胞增生李斯特氏菌(Listeria monocytogenes,以下简称单增李斯特氏菌)是生食三文鱼产品中的高风险致病微生物。为探明生食三文鱼产品中单增李斯特氏菌的菌株特性及其基因组特征与分子型别、毒力基因与耐药基因携带等情况。本研究对分离自生食三文鱼产品中的1株ST18型单增李斯特氏菌LMSUR02进行细胞毒性实验、药敏实验与血清型鉴定等表型实验,并对其进行全基因组测序。借助相关软件对测序数据进行预测与注释,并对LMSUR02与来自其他不同地区和年份的单增李斯特氏菌分离株进行比较基因组分析。结果表明,LMSUR02的细胞毒性显著高于标准菌株ATCC19115,对头孢呋辛钠、头孢曲松、头孢吡肟与萘啶酸等药物耐受,其血清型为I.1(1/2a,3a);LMSUR02基因组大小为2945085 bp,在其基因组中共预测到1个CRISPR序列、3个基因组岛与2个前噬菌体;LMSUR02来自进化谱系II,属于CC18克隆系ST18序列型,携带LIPI-1(Listeria pathogenicity island 1)与LIPI-2(Listeria pathogenicity island 2)两个主要的毒力岛及耐药基因norB、lin与FosX;LMSUR02与IZSAM_Lm_15_17439_A144(GCA_001513695.1)、Lm_N1546(GCA_001483445.1)、HPB913(GCA_001913195.1)、LM26(GCA_016757715.1)、J2-031(GCA_000438645.1)、2015TE19005-1355(GCA_001551855.1)、B-33260(GCA_016801005.1)、FW040025(GCA_001644625.1)及Lm_3136(GCA_001483425.1)位于同一进化分支且10株单增李斯特氏菌的共线性较好,其中以Lm_3136(GCA_001483425.1)与LMSUR02的亲缘关系最近。本研究明晰了1株ST18型单增李斯特氏菌的菌株特性,丰富了单增李斯特氏菌的基因组数据库,为防控因生食三文鱼等生食水产品导致的单增李斯特氏菌感染提供重要理论基础。

    Abstract:

    Listeria monocytogenes is a significant foodborne pathogen responsible for zoonotic diseases, which is considered one of the most important contaminants in raw aquatic products. Infection with L. monocytogenes can cause listeriosis, a serious disease primarily affecting pregnant women, newborns, the elderly and other immunocompromised individuals, with a high mortality rate of 20-30%. Salmon, as an important raw aquatic product, is a high-risk food that is often contaminated with L. monocytogenes. On the one hand, raw salmon products are often transported and stored at low temperatures, but L. monocytogenes can still survive at refrigerated conditions. On the other hand, raw salmon products are directly consumed without heat treatment, which cannot effectively inactivate L. monocytogenes that may be present. Therefore, there is a significant risk of consumers being infected with L. monocytogenes. In recent years, there have been multiple outbreaks of listeriosis caused by raw salmon products worldwide, resulting in severe health damage and economic losses. ST18 L. monocytogenes has been reported to be associated with the outbreak of listeriosis, however, there are few reports on the ST18 strains. Thus, it is important to clarify the characteristics and genetic diversity of ST18 L. monocytogenes in raw salmon products, which is of great significance for the prevention and control of listeriosis, and enhancing the food safety of raw salmon products. Nowadays, whole-genome sequencing (WGS) technology has become a powerful tool to investigate L. monocytogenes outbreaks due to its high efficiency, accuracy and comprehensive genomic data. To elucidate the characteristics, genomic features and molecular types as well as the virulence genes and antimicrobial resistance genes distribution of ST18 L. monocytogenes in raw salmon products. Phenotypic characterization, including cytotoxicity assay, antimicrobial susceptibility testing and serotyping of L. monocytogenes strain LMSUR02 isolated from raw salmon products were performed. The cytotoxicity and antimicrobial susceptibility of the isolates were evaluated by determining the content of lactate dehydrogenase released by Caco-2 cells and disc diffusion method, respectively, and the serogroups were identified by multiple PCR technology. Then WGS of LMSUR02 was subsequently conducted. The sequencing data were predicted and annotated by using relevant software, and comparative genomic analysis with other L. monocytogenes isolates from different regions and years were conducted. The results indicated that LMSUR02 exhibited significantly higher cytotoxicity of 66.53% than the reference strain ATCC19115. LMSUR02 contains two major Listeria pathogenicity islands (LIPI-1 and LIPI-2), LIPI-3 and LIPI-4 were absent. LMSUR02 was susceptible to most of the antimicrobial tested, showed intermediary to ofloxacin and ciprofloxacin, and displayed resistance to cefuroxime sodium (CXM), ceftriaxone (CRO), cefepime (FEP) and nalidixic acid (NA). The strain carried antimicrobial resistance genes norB, lin and FosX. By comparing the antimicrobial resistance phenotype with the genotype, it was found that LMSUR02 is resistant to CXM, CRO, FEP and NA, but corresponding resistance genes were not detected. This suggests that non-specific efflux mechanisms might contribute to their resistance. Serotyping classified LMSUR02 as serotype I.1 (1/2a, 3a). The sporadic and outbreak cases of listeriosis are predominantly caused by serotypes 4b and 1/2a. Given the increasingly strong association between serotype 1/2a and listeriosis, it is suggested that LMSUR02 has a relatively high risk of causing listeriosis and should be taken seriously. The assembled genome of LMSUR02 was 2945085 bp, harboring one CRISPR sequence, three genomic islands and two prophages. LMSUR02 belonged to sequence type CC18 (ST18). In previous studies, the CC18 stains were detected in both environmental and clinical isolates, indicating that LMSUR02 has a potential risk of inducing listeriosis, which should be given high attention. L. monocytogenes strains can be phylogenetically categorized four evolutionary lineages (I, II, III, and IV), with lineage II being the most prevalent among isolates from foods and environmental sources. LMSUR02 originated from evolutionary lineage II, consistent with previously reported findings. Comparative genomics indicated a close evolutionary relationship between LMSUR02 and nine other stains IZSAM_Lm_15_17439_A144 (GCA_001513695.1), Lm_N1546 (GCA_001483445.1), HPB913 (GCA_001913195.1), LM26 (GCA_016757715.1), J2-031 (GCA_000438645.1), 2015TE19005-1355 (GCA_001551855.1), B-33260 (GCA_016801005.1), FW040025 (GCA_001644625.1) and Lm_3136 (GCA_001483425.1) with strong genomic synteny among all ten strains. The genomic module compositions of the ten strains were similar, all containing eight collinear modules, but the specific positions and arrangement sequences of these modules in the genome were different. Among nine stains, Lm_3136 (GCA_001483425.1) has the closest evolutionary relationship with LMSUR02. In summary, this study clarified the characteristics of a ST18 L. monocytogenes strain from raw salmon products and enriched the genome database of L. monocytogenes, which provides an important theoretical basis for the prevention and control of L. monocytogenes infection associated with raw aquatic products including raw salmon products.

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  • 收稿日期:2025-09-02
  • 最后修改日期:2025-09-30
  • 录用日期:2025-10-09
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