A Filamentous Phage Associated with Recent Pandemic Vibrio parahaemolyticus O3:K6 Strains

Abstract

A specific serotype, O3:K6, of Vibrio parahaemolyticus has recently been causing epidemics of gastroenteritis in Southeast Asia, Japan, and North America. To examine whether the new O3:K6 strains possess characteristics that may exacerbate outbreaks, we compared V. parahaemolyticus O3:K6 strains with non-O3:K6 strains using strains isolated from individuals with traveler's diarrhea at Kansai Airport Quarantine Station, Osaka, Japan. All 24 O3:K6 strains possessed a common plasmid, pO3K6 (DNA size, 8,782 bp, with 10 open reading frames [ORFs]). The gene organization of pO3K6 was similar to that of Vf33, a filamentous phage previously described in V. parahaemolyticus. We isolated a phage (phage f237) from the culture supernatant of V. parahaemolyticus O3:K6 strain KXV237, which formed a turbid plaque on an indicator strain. The genome of f237 was single-stranded DNA, and the double-stranded DNA obtained by treatment of the genome with DNA polymerase was identical to that of pO3K6 when analyzed by agarose gel electrophoresis after HindIII digestion. Furthermore, the N-terminal amino acid sequence of the f237 major coat protein was found in ORF4 of pO3K6. Our results showed that pO3K6 is a replicative form of f237. Among the ORFs found in the f237 genome, the sequence of ORF8 had no significant homology to those of any proteins in databases. ORF8 was located on a region corresponding to the distinctive region of Vf33, and its G؉C content was apparently lower than that of the remaining DNA sequence of f237. By colony hybridization, ORF8 was detected only in O3:K6 strains isolated since 1996 and was not found in O3:K6 strains isolated before 1996 and clinical V. parahaemolyticus strains other than those of serotype O3:K6. Thus, this study shows that f237 is exclusively associated with recent V. parahaemolyticus O3:K6 strains. The ORF8 gene can be a useful genetic marker for the identification of the recently widespread O3:K6 strains of V. parahaemolyticus.

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

“…Previous studies demonstrated a strong association of filamentous phages, f237, with recent pandemic strains of V. parahaemolyticus. This phage possesses a unique ORF8 open reading frame that is found only in recent pandemic isolates, and the Zot (Zonula occludens toxin)-like toxin previously described in the V. cholerae is reported to be encoded in the ORF7 [51,52]. A match was found between the CRISPR spacer region of V. anguillarum PF7 with a zot-encoding prophage in V. anguillarum PF4, indicating the contribution of CRISPR to the phage resistance of this bacterium [53].…”

Beyond Cholera: Characterization of zot-Encoding Filamentous Phages in the Marine Fish Pathogen Vibrio anguillarum

“…Recent studies predicted the zot-like genes to reside on prophage elements in three V. anguillarum strains isolated from Chile, the UK and the US, respectively [26], and suggested that the loss of a zot-encoding prophage reduced virulence against fish larvae in a pathogenic V. anguillarum [27]. These results suggest that Inovirus-related prophages may spread in V. anguillarum and that inoviruses could contribute to the virulence of this bacterium, as proposed for the related species V. cholerae and V. parahaemolyticus [13,14,23,28].…”

“…Sequencing of the putative replicative forms revealed two novel Inovirus genomes, pVAI1 and pVAI2. The genomes of pVAIs consisted of 6117 bp, were predicted to encode 11 ORFs, had a GC content of 42.5% and contained two intergenic regions corresponding to conserved sequences of inoviruses [13,28]. Global alignment of the genomes of pVAI1 and pVAI2 exhibited a pairwise identity of 99.95%.…”

Whole genome analysis unveils genetic diversity and potential virulence determinants in Vibrio parahaemolyticus associated with disease outbreak among cultured Litopenaeus vannamei (Pacific white shrimp) in India

“…ORFs from f237 were found among the identified prophage sequences. The phage f237 has been linked to pandemic clones since 1996, and is thought to play a key role in increasing bacterial virulence [ 53 ]. Furthermore, VP32 had prophage sequences from the Myoviridae family, while the HP1 and SHP/2 genomes contained prophage sequences from the Myoviridae and Siphoviridae families.…”

“…Furthermore, VP32 had prophage sequences from the Myoviridae family, while the HP1 and SHP/2 genomes contained prophage sequences from the Myoviridae and Siphoviridae families. These phages are members of the order Caudovirales, which includes marine bacteriophages known to infect Vibrio sp [ 53 , 54 ]. Apart from being acquired horizontally, some of these prophages could have integrated into the sequenced bacterial genome and may have been transferred vertically through generations [ 55 ].…”