Vibrio parahaemolyticus is a halophilic gram-negative bacterium widely found in aquatic environments, including estuarine, marine, and coastal ecosystems (Ceccarelli et al., 2013). This species is a leading causative agent of foodborne human acute gastroenteritis, which can be transmitted by the consumption of uncooked, undercooked, or mishandled marine products (Newton et al., 2012). Specific antigen types of virulent V. parahaemolyticus cause several life-threatening infections and large food poisoning outbreaks. Moreover, most virulent isolates have acquired resistance to several antimicrobial agents, which could be an important reservoir for various antimicrobial resistance (AMR) genes (Letchumanan et al., 2014). Therefore, V. parahaemolyticus poses a risk to public health and food safety.

Moreover, V. parahaemolyticus is the major etiological agent of acute hepatopancreatic necrosis disease (AHPND or early mortality syndrome), which causes significant economic losses in the global shrimp aquaculture industry. The AHPND-causing V. parahaemolyticus (Vp_AHPND), which produces the PirAB toxin, is characterized by the presence of a large (~70 kb) conjugative plasmid (pVA1) containing two toxin subunit genes (pirA and pirB) (Lee et al., 2015). Since its first outbreak in China in 2009, AHPND has been reported in several countries in the Americas and Asia, including Korea, and has caused serious economic losses up to recently (Han et al., 2020). Previously, we reported the first complete genome of the pVA1-type plasmid (pVp_Kor-D1-2) of Vp_AHPND 19-021-D1, which caused a severe AHPND outbreak in 2019 among cultured shrimp in Korea (Han et al., 2020). In this study, we present the whole genomic characteristics of the bacterial chromosomes and plasmids of Vp_AHPND 19-021-D1.

In our previous study, Vp_AHPND 19-021-D1 was isolated from the culture pond water of pacific white shrimp (Penaeus vannamei) with AHPND (Han et al., 2020). The total genomic DNA of Vp_AHPND 19-021-D1 was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and sequenced via a hybrid approach: (1) using the PacBio RSII System (Pacific Biosciences) by constructing a 20-kb SMRTbell^TM template library and (2) using the HiSeq X-10 platform (Illumina) by preparing a DNA library using the TruSeq Nano DNA Library Prep Kit (Illumina). The PacBio reads (1,387,556,996 bp; 141,754 reads) were de novo assembled with the HGAP (v3.0) (Chin et al., 2013). Then, the Illumina paired-end reads (898,634,220 bp, 5,951,220 reads) were mapped to the PacBio assembled genome using BWA-MEM v0.7.17 (Li and Durbin, 2009), and the final error correction was conducted with Pilon v1.21 (Walker et al., 2014). Genome annotation was performed using the NCBI prokaryotic genome annotation pipeline (http://www.ncbi.nlm.nih.gov/books/NBK174280/).

The sequenced genome of strain 19-021-D1 consisted of two circular chromosomes (encoding 4,923 CDS, 133 tRNAs, 37 rRNAs, and four ncRNAs) and two plasmids (pVp_Kor-D1-1 and pVp_Kor-D1-2) (encoding 200 CDS). Chromosome I was 3,577,848 bp in length (145× coverage) with 45.15% G + C content. Chromosome II was 1,814,246 bp in length (132× coverage) with 45.65% G + C content. The plasmid pVp_ Kor-D1-1 was 118,438 bp in length (310× coverage), with 44.29% G + C content. Lastly, the pVA1-type plasmid pVp_ Kor-D1-2 was 68,848 bp in length (176× coverage) with 45.81% G + C content (Fig. 1 and Table 1).

Fig. 1. The genome map of Vibrio parahaemolyticus 19-021-D1.
A circular graphical genome map (A) of strain 19-021-D1 and its subsystems (B) were respectively annotated and generated by using the Pathosystems Resource Integration Center (PATRIC) server v.3.6.12 (Wattam et al., 2017).

Table 1 The general genome features of AHPND-causing V. parahaemolyticus strain 19-021-D1

Features	Value
Genome size (bp)	5,579,380
Chromosome I	3,577,848
Chromosome II	1,814,246
Plasmid (pVp_Kor-D1-1)	118,438
Plasmid (pVp_Kor-D1-2)	68,848
G + C content (%)	45.09
No. of contigs	4
Plasmid	2
Total genes	5,297
Protein-coding genes	5,052
rRNAs	37
tRNAs	133
ncRNAs	4
Pseudogenes	71

Potential virulence-associated genes in the 19-021-D1 strain were compared with those of V. parahaemolyticus strains available in the GenBank database and additional virulence-and AMR-related genes (ARGs) were screened as previously described (Lee et al., 2018). Among the three major virulence factors in this species, the 19-021-D1 genome possessed only thermolabile hemolysin (TLH) on chromosome II. On the other hand, thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are responsible for human gastroenteritis (Liston, 1990), were not detected (Supplementary data Table S1). Although the Vp_AHPND 19-021-D1 showed no significant phenotypical resistance against several antimicrobial agents of the guidelines of the Clinical and Laboratory Standards Institute (Han et al., 2020), several ARGs were detected by Resistance Gene Identifier on the criteria of ‘perfect and strict’ in the Comprehensive Antibiotic Resistance Database (Alcock et al., 2020) in the chromosomes of strain 19-021-D1. Overall, the ARGs in strain 19-021-D1 were very similar to those reported in V. parahaemolyticus Vp2015094, which was isolated from maricultured shellfish in China (Wang et al., 2022); (i) three efflux pump genes, adeF (fluoroquinolone and tetracycline resistance), crp (macrolide, fluoroquinolone, and penam resistance), and txR (tetracycline resistance); and (ii) the antibiotic inactivation gene bla_CARB-18 (penam resistance). However, one antibiotic target alteration gene, vanT (glycopeptide antibiotic resistance), was detected only in strain 19-021-D1. Additionally, none of the ARGs was detected in the two plasmids (Supplementary data Table S2).

Eight prophage regions were identified in the 19-021-D1 chromosome (six in chromosome I and two in chromosome II) using the Phage Search Tool Enhanced Release (PHASTER) server (Arndt et al., 2016), one of which was intact (score > 90), and five were incomplete (score < 70). The only intact prophage region of the 19-021-D1 genome was detected in chromosome II consisted of 11.6 kb (Region 8) and showed significant similarity with Vibrio phage vf33 (NC_005948) and CTX (NC_015209) (Supplementary data Table S3).

Moreover, we performed multilocus sequence typing (MLST) analyses to determine the sequence type (ST) of Vp_AHPND 19-021-D1. A total of seven housekeeping gene (dnaE, gyrB, recA, dtdS, pntA, pyrC, and tnaA) sequences of the sequenced genome were compared in silico with those available in the PubMLST database (https://pubmlst.org/organisms/vibrio-spp) and its respective allele numbers and ST were generated. The allele profile of the Vp_AHPND 19-021-D1 was 47-8-166-19-28-46-121, and its ST was finally assigned to 413. Interestingly, we found six other strains of ST 413 V. parahaemolyticus in the PubMLST database, all of which were isolated from diseased or unknown shrimp products of different geographical origins (including South Korea, China, Thailand, Vietnam, and the USA) (Supplementary data Table S4).

Therefore, we conducted the genome-based comparison of the strain 19-021-D1 against the available genomes of three ST 413 strains and other three Vp_AHPND strains using the OAT software (Lee et al., 2016). The results indicated that the chromosomes of strain 19-021-D1 were clustered with all the tested V. parahaemolyticus strains, and the four ST 413 strains were nested together within the clade showing high orthologous average nucleotide identity values (≥ 99) (Supplementary data Fig. S1). Although detailed information on diseased shrimp samples is not currently available, it can be speculated that ST 413 V. parahaemolyticus is strongly associated with culturing shrimps, and some strains might be involved in the onset of the disease in shrimp. However, further research is required to verify the potential relationship between ST 413 V. parahaemolyticus and AHPND. Based on the genetic characterization of Vp_AHPND that occurred in Korea in 2019, we confirmed that this shrimp-specific pathogen could pose a threat to public health including food safety due to the presence of TLH. These results provide important insights for the development of strategies to control Vp_AHPND which has caused serious economic losses in the Korean shrimp culture industry. This is the first genome report of Vp_AHPND in Korea, and further genome-based investigation of recent Korean Vp_AHPND isolates is urgently required to ensure the safety of shrimp products.

Nucleotide sequence accession number

The complete genome sequences of Vp_AHPND 19-021-D1 have been deposited in the GenBank database under accession numbers NZ_CP046411.1 (chromosome I), NZ_CP046412.1 (chromosome II), NZ_CP046413.1 (pVp_Kor-D1-1), and NZ_CP046414.1 (pVp_Kor-D1-2).

수산물 유래 식중독 세균인 Vibrio parahaemolyticus는 전세계 공중보건과 식품위생에 심각한 위해를 야기하고 있다. 더불어, pirA 및 pirB 독성 유전자를 암호화하는 플라스미드(pVA1)를 보유하는 특정 V. parahaemolyticus 균주는 양식 새우의 급성간췌장괴사증(AHPND)을 유발하여 전세계 새우 양식산업에 심각한 경제적 손실을 야기하는 주 병원체로 알려져 있다. 이전 연구에서, 2019년도에 최초로 국내 양식 흰다리새우(Penaeus vannamei)에서 분리한 AHPND 발병 균주, V. parahaemolyticus 19-021-D1의 플라스미드(pVA1) 완전 유전체를 보고한 바 있으며, 본 연구에서는 19-021-D1 균주의 전장유전체 분석을 통해 병원성 및 항생제 내성 관련 유전적 특성을 분석하였다. 분석 결과, 19-021-D1 균주는 두 개의 원형 염색체와 두 개의 플라스미드로 구성되며, 병원성, 항생제 내성, prophage region과 관련이 있을 것으로 추정되는 다양한 유전자가 검출되었다. 다좌위 서열 형별 분석(MLST) 결과 19-021-D1 균주는 413타입으로 분류되었으며, 흥미롭게도, 기존 다른 국가의 양식 새우에서 분리된, 질병과 연관된 것으로 추정되는 균주들과 동일한 타입으로 분류되었다. 이러한 연구 결과는 국내 수산양식 산업 및 식품 공중보건 분야에서 문제를 야기하는 V. parahaemolyticus를 이해하고 제어하기 위한 중요한 기반자료로 활용될 것이다.

This research was supported by the National Research Foundation of Korea (NRF-2020R1I1A2068827) funded by the Ministry of Education, and by the Development of Technology for the Biomaterialization of Marine Fishery Byproducts of the Korea Institute of Marine Science & Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (KIMST-20220128).

All authors disclose no conflicts of interest relevant to this research.