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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(3): 328-330

https://doi.org/10.48022/mbl.2406.06006

Received: June 13, 2024; Revised: July 26, 2024; Accepted: July 31, 2024

Whole-Genome Analysis of CC224 Listeria monocytogenes Strain IJPL9-1, Clonally Related to the Listeriosis Outbreak Strain in 2018, Isolated from Pork in Korea

Mi Ru Lee and Kun Taek Park*

Department of Biological Sciences, Inje University, Gimhae 50834, Republic of Korea

Correspondence to :
Kun Taek Park,        ktpark@inje.ac.kr

Listeriosis is one of serious foodborne disease caused mainly by consumption of food contaminated with Listeria monocytogenes. In this study, we isolated L. monocytogenes strain IJPL9-1 from pork in Korea and conducted whole-genome sequencing (WGS). WGS data revealed a single chromosome of 2,913,085 bp. The strain was identified as sequence type (ST) 224, clonal complex (CC) 224, lineage I, and sub-lineage (SL) 6178 based on multilocus sequence typing (MLST) and core genome MLST (cgMLST). The average nucleotide identity was 95.15% with the reference genome EGD-e and 99.99% with FSCNU_000110, the outbreak strain in Korea in 2018. The serogroup was determined to be IIb, and the presence of antimicrobial resistance genes fosX, vga(G), mprF, norB, and sul was determined.

Keywords: Listeria monocytogenes, whole-genome sequence, clonal complex 224, Pork

Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis, a type of food poisoning in humans. Approximately 1,600 people are diagnosed with listeriosis annually, resulting in 260 fatalities. Listeriosis is more dangerous in pregnant women and their newborns, adults aged > 65 years, and individuals with weakened immune systems [1].

In South Korea, the first foodborne outbreak in 2018 was associated with seasoned crab meat with bean sprouts served in school lunches. This outbreak resulted in 64 confirmed cases of L. monocytogenes infection [2]. The causative strain (FSCNU0110) was identified as multi-locus sequence type (MLST)-based sequence type of 224 (ST224), clonal complex type of 224 (CC224), and a core genome MLST (cgMLST) sub-lineage (SL) type of 6178 (SL6178), which is a distinct domestic strain different from ST224 strains reported in other countries [3].

In this study, strain IJPL9-1 was isolated from pork sold in a domestic supermarket, subjected to MLST analysis, and was identified as ST224. Due to the same ST of the previous outbreak strain (FSCNU0110) in 2018, we performed whole-genome sequencing (WGS) analysis to reveal the clonal relatedness. Genome sequencing was conducted using Illumina NextSeq 2,000 and Nanopore platforms. DNA was extracted using a NucleoSpin Microbial DNA Mini Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol. For NextSeq 2,000 sequencing, libraries were prepared using a TruSeq Nano DNA Prep kit (Illumina, USA) in accordance with the manufacturer’s instructions. The libraries were sequenced using NextSeq P1 600 cycles in a NextSeq2000 system using 2 × 300 bp paired-end reads. After sequencing, individual sequence reads were analyzed using FastQC (v0.11.8). For Nanopore sequencing, libraries were prepared using a Ligation Sequencing kit (Oxford Nanopore Technologies). Nanopore sequencing data were processed using Guppy (v4.2.2). Illumina and Nanopore sequencing data were processed and hybridized using Unicycler (v0.5.0), and the genome was annotated using Prokka (v1.14.6). The high-quality complete genome sequence of IJPL9-1 was deposited in the National Center for Biotechnology Information (NCBI) GenBank database (NCBI accession number CP157376).

The assembled IJPL9-1 genome comprised a single chromosome with a length of 2,913,085 bp and a GC content of 38.01%. The genome contained 2,799 coding sequences (CDS), 18 rRNA genes, and 67 tRNA genes (Table 1 and Fig. 1). For comparison with a reference strain and FSCNU0110 genomes, average nucleotide identity (ANI) was calculated using FastANI (v1.3.3) [4]. The ANI value with the NCBI reference genome EGD-e (NC_003210.1) was 95.15% (Fig. 2A). A comparison with the previously reported outbreak strain genome FSCNU0110 (CP101619.1) [3] revealed a 99.99% identity (Fig. 2B).

Table 1 . Genetic characteristics of L. monocytogenes IJPL9-1.

FeaturesCharacteristics
No. of contig1
Genome size2,913,085 bp
GC content38.01%
No. of CDS2,799
No. of rRNA18
No. of tRNA67
MLSTST224, CC224, lineage I
cgMLST Type, sub-lineageNot typeable, SL6178
SerogroupIIb
Antimicrobial resistance genefosX, vga(G), mprF, norB, sul

Notes: CDS, coding sequence; MLST, multi-locus sequence typing; ST, sequence type; CC, clonal complex; SL, sub-lineage



Figure 1.Circular map of the L. monocytogenes IJPL9-1 chromosome. The features from the outside to the inside are CDS on the forward strand, CDS on the reverse strand, tRNA, rRNA, G+C content, and GC skew.

Figure 2.Comparison of the average nucleotide identity (ANI) with other genomes. (A) 95.51% similarity with the NCBI reference genome EGD-e. (B) 99.99% similarity with the genome from the outbreak strain FSCNU00110.

Based on the WGS data of IJPL9-1, the MLST was identified using mlst (v2.23.0) [5] with the BIGSdb_LM scheme for seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and lhkA) [6], which was classified as ST224, CC224, and lineage I. cgMLST analysis of BIGSdb_LM (1,748 allele profiles) [7] identified a sub-lineage (SL) with up to 150 allele differences, designated as SL6178. However, no cgMLST types with up to seven allele differences were identified.

The serogroup was identified to be IIb using LisSero (v0.4.9) [8]. To identify antimicrobial resistance genes, AMRFinderPlus (v3.11.26) and BIGSdb_LM were used, which revealed the presence of fosX (fosfomycin), vga(G)(lincosamides), mprF (cationic peptides), norB (quinolones), and sul (sulfonamides) (Table 1). Compared to FSCNU0110, IJPL9-1 strain lost tetM (tetracycline) resistant gene in the genome.

The results of a previous [3] and this studies suggest a clonal distribution of distinct lineage of L. monocytogenes in different food sources in Korea, which emphasize the need of surveillance program to manage the risk of L. monocytogenes contamination in domestic food production and processing environments.

This study was supported by a grant from the Ministry of Food and Drug Safety (22192MFDS021) of the Republic of Korea.

The authors have no financial conflicts of interest to declare.

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