New Sequence Types of Vibrio parahaemolyticus Isolated from a Malaysian Aquaculture Pond, as Revealed by Whole-Genome Sequencing

ABSTRACT

The acquisition of Photorhabdus insect-related (Pir) toxin-like genes in Vibrio parahaemolyticus has been linked to hepatopancreatic necrosis disease in shrimp. We report the whole-genome sequences of genetically virulent and avirulent V. parahaemolyticus isolated from a Malaysian aquaculture pond and show that they represent previously unreported sequence types of V. parahaemolyticus.

GENOME ANNOUNCEMENT

Vibrio parahaemolyticus is a marine Gram-negative bacterium (1) that has been occasionally associated with acute hepatopancreatic necrosis disease (AHPND) in the white leg shrimp, Litopenaeus vannamei, resulting in severe economic losses in shrimp production in Southeast Asian countries (2). Photorhabdus insect-related (Pir) toxin-like genes have been recently identified in various AHPND-causing V. parahaemolyticus strains and these genes (pirA- and pirB-like) were shown to be the primary virulence factor in these strains (3).

Five V. parahaemolyticus strains (MVP1, MVP2, MVP4, MVP6, and MVP9) were isolated from a shrimp pond that was tested positive for V. parahaemolyticus harboring the Pir genes. Genomic DNA was extracted from a 2-day-old marine nutrient agar culture (ATCC Medium 8) using the Solokov method (4). Library preparation was performed using the NexteraXT DNA library preparation kit (Illumina, San Diego, CA) according to the manufacturer’s instructions and sequenced on a MiSeq desktop sequencer (2 × 75-bp and 2 × 250-bp configurations) located at the Monash University Malaysia Genomics Facility.

Nextera adapter trimming was performed using Trimmomatic version 0.32 (5) and the filtered paired-end reads were assembled using SPAdes version 3.8.1 (6). After the removal of short (<300 bp) and/or low-coverage (<2×) contigs, in silico scaffolding and gap closing were performed using SSPACE version 2.1 (7) and Gapfiller version 1.10 (8), respectively. To confirm the identity of the isolated strains as V. parahaemolyticus, Jspecies version 1.2 (9) was used to calculate the average nucleotide identity of strains MVP1, -2, -4, -6, and -9 in comparison to the whole genome of V. parahaemolyticus DSM 10027^T. Subsequently, gene prediction was performed using Prodigal version 2.6 (10) and searched against the multilocus sequence typing (MLST) locus database (http://www.mlst.net/) to infer the sequence type of each sequenced strain based on their genetic similarity to seven housekeeping genes, namely, pyrC, gyrB, recA, dnaE, tnaA, pntA, and dtdS. The identification of the Pir genes was performed via a local BlastN search against pirA (GenBank accession no. AIL49948.1) and pirB (GenBank accession no. AIL49949.1).

A summary of the assembly statistics for the genomes of all isolates is available in Table 1. All five strains exhibited more than 95% average nucleotide identity (ANI) to the type strain of V. parahaemolyticus. Based on the lack of 100% sequence identity to seven housekeeping genes, new MLST sequence types of V. parahaemolyticus were identified in this study (Table 1). Subsequent blastN searches showed that strains MVP1, MVP2, and MVP6 contain the identical nucleotide sequence for all seven housekeeping genes (Table 1, data not shown), thus classifying them as the same sequence type. In addition to sharing the identical sequence type, these 3 strains also harbor the Pir toxin genes. On the contrary, strains MVP4 and MVP9 belong to two different sequence types and do not harbor the Pir toxin genes, suggesting a potential association between the sequence types and the presence of Pir toxin genes in V. parahaemolyticus.

TABLE 1.

Accession numbers and genetic analyses of V. parahaemolyticus strains reported in this study

Strain	Accession no.	Genome size (bp)	N50 (bp)	No. of contigs	pyrCa	gyrBa	recAa	dnaEa	tnaAa	pntAa	dtdSa	Pirb
MVP1	MQMQ01000000	5,230,330	60,033	172	303	143c (591/592)	218	308c (556/557)	26	30	355c (457/458)	+
MVP2	MSBY01000000	5,275,177	129,821	89	303	143c (591/592)	218	308c (556/557)	26	30	355c (457/458)	+
MVP4	MSBZ01000000	5,270,749	94,277	122	27	141	31c (728/729)	110c (556/557)	26	18	232	−
MVP6	MSCA01000000	5,195,990	47,800	190	303	143c (591/592)	218	308c (556/557)	26	30	355c (457/458)	+
MVP9	MSCB01000000	4,967,664	87,871	115	54c (492/493)	144	116	28	61	26	252	−

^aNumerical values indicate the MLST allele for the respective genes.

^b+, presence of both pirA and pirB genes; −, absence of both pirA and pirB genes.

^cClosest allele hit, with values in parentheses indicating the number of positions over the length of the gene fragment where all of the bases at that position are identical.

Accession number(s).

This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession numbers listed in Table 1. The versions described in this paper are the first versions.