PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

Vibrio parahaemolyticus is a natural inhabitant of coastal waters worldwide (6, 25). This organism is a halophilic, gram-negative bacterium that causes gastroenteritis in humans. Infection results from the consumption of contaminated seafood, particularly raw shellfish. A significant increase in the number of cases of V. parahaemolyticus infections was reported in 1996. A unique clone of V. parahaemolyticus O3:K6 is responsible for many of the recent V. parahaemolyticus outbreaks, including epidemics in India, Russia, Southeast Asia, Japan, and North America (9, 13, 17, 23). Strains of the O3:K6 serovar emerged in Calcutta, India, in 1996 and have accounted for 50 to 80% of V. parahaemolyticus infections annually since then. The same serovar was isolated from patients in various Southeast Asian countries, and it was found that the O3:K6 strains isolated since 1996 are all derived from a single clone (20). In 1997, an outbreak caused by V. parahaemolyticus occurred in the Pacific Northwest, resulting in one death and 209 illnesses. Each of the infected persons had consumed raw oysters (8). V. parahaemolyticus O3:K6 was involved in outbreaks from contaminated oysters harvested from Oyster Bay, N.Y., and Galveston Bay, Tex., in 1998 (8, 12). The V. parahaemolyticus outbreak in Galveston Bay was the largest reported in the United States (416 persons), and all clinical isolates were the O3:K6 serotype (12). It was determined that a filamentous phage, f237, is specifically and exclusively associated with O3:K6 serovar strains isolated since 1996 (19). This phage has 10 open reading frames (ORFs), including a unique open reading frame, ORF8, which shows no homology to known DNA sequences. Vibrio cholerae has a similar filamentous phage, CTX, which carries the cholera enterotoxin genes ctxA and ctxB (15). In V. parahaemolyticus O3:K6, the f237 phage has ORF8 in place of ctxAB (26). Therefore, it is suggested that ORF8 may play a role in the virulence of the strains that possess it because of the increased infection rates in these strains (19, 20, 22). The “new” V. parahaemolyticus O3:K6 pathogenic strain with the f237 phage was first isolated in Calcutta, India. Like the spread of the epidemic strain of V. cholerae O1 in 1991 to coastal waters along the Gulf of Mexico (18), it is probable that the spread of the pathogenic V. parahaemolyticus O3:K6 strain occurred via ship ballast water, which is believed to be the leading cause for the spread of nonindigenous organisms in the marine environment. Therefore, it is necessary and desirable to be able to detect the pathogenic strains of V. parahaemolyticus O3:K6 in the environment, particularly in marine water, to monitor its presence. Detection of V. parahaemolyticus O3:K6 in waters where shellfish are harvested can help prevent infections resulting from ingestion and thus improve the safety of shellfish. This detection will benefit the shellfish industry and will ensure consumer confidence in the consumption of seafood. To prevent infections, it is necessary to detect this pathogen rapidly and reliably. Conventional biochemical methods for the detection of V. parahaemolyticus are time-consuming, requiring several days to acquire confirmatory results (14, 16). Several gene-based methodologies that target species and virulence gene segments, like PCR (11, 24; G. Blackstone, Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001, abstr. Q-454, p. 676, 2001), colorimetric DNA-DNA hybridization (18), and multiplex PCR (4), have been developed. However, these methodologies are not specific for the detection of newly emerged pathogenic V. parahaemolyticus O3:K6 strains. In this study, we describe the selection of ORF8 template-specific oligonucleotide primers and the optimization of PCR amplification to establish a qualitative PCR with high specificity and sensitivity for the detection of these newly emerged pandemic isolates of V. parahaemolyticus O3:K6 in pure cultures and in seeded gulf waters.