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.
