Characterization and Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Pandemic and Non-Pandemic Vibrio parahaemolyticus Isolates from Seafood Sources.

Nawaporn Jingjit,Komwit Surachat,Sutima Preeprem,Pimonsri Mittraparp-Arthorn

doi:10.3390/microorganisms9061220

Abstract

Vibrio parahaemolyticus is one of the significant seafood-borne pathogens causing gastroenteritis in humans. Clustered regularly interspaced short palindromic repeats (CRISPR) are commonly detected in the genomes of V. parahaemolyticus and the polymorphism of CRISPR patterns has been applied as a genetic marker for tracking its evolution. In this work, a total of 15 pandemic and 36 non-pandemic V. parahaemolyticus isolates obtained from seafood between 2000 and 2012 were characterized based on hemolytic activity, antimicrobial susceptibility, and CRISPR elements. The results showed that 15/17 of the V. parahaemolyticus seafood isolates carrying the thermostable direct hemolysin gene (tdh+) were Kanagawa phenomenon (KP) positive. The Multiple Antibiotic Resistance (MAR) index ranged between 0.1 and 0.4, and 45% of the isolates have an MAR index ≥ 0.2. A total of 19 isolates were positive for CRISPR detection, including all tdh+ trh− isolates, two of tdh− trh+, and each of tdh+ trh+ and tdh− trh−. Four spacer types (Sp1 to Sp4) were identified, and CRISPR-positive isolates had at least one type of spacer homolog to the region of Vibrio alginolyticus megaplasmid. It is of interest that a specific CRISPR profile and spacer sequence type was observed with correlations to the hemolysin genotype (tdh/trh). Thus, these provide essential data on the exposure of foreign genetic elements and indicate shared ancestry within different genotypes of V. parahaemolyticus isolates.

Highlights

Introduction iationsVibrio parahaemolyticus is a Gram-negative halophilic bacterium which belongs to the family Vibrionaceae
Isolated from seafood sources were grouped based on the presence of tdh, trh, and the pandemic marker genes detected grouped based on the presence of tdh, trh, and the pandemic marker genes detected by by
This study found that the sequences of Clustered regularly interspaced short palindromic repeats (CRISPR) repeats found among V. parahaemolyticus isolates were not highly identical, especially at the terminal position, this does not affect the RNA secondary structures

Summary

Introduction

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium which belongs to the family Vibrionaceae. It is an oxidase-positive, facultative anaerobic bacterium, similar to other members in the genus Vibrio, present in marine or estuarine environments [1]. Many V. parahaemolyticus strains are pathogenic and can cause gastroenteritis in humans due to consumption of raw or undercooked seafood [2]. It was discovered as a common cause of foodborne diseases in Japan in 1950 and is responsible for the world’s worst seafood-associated diarrhoea after new pandemic strains emerged in 1996 [3]. The virulence genes, tdh and trh, encoded for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), respectively, are considered as virulence factors associated with V. parahaemolyticus hemolysis and cytotoxicity activity in the host, and have been used as pandemic group-specific markers together with group-specific toxRS PCR (GS-PCR) to Licensee MDPI, Basel, Switzerland.

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