Molecular characterization of Vibrio parahaemolyticus strains associated with foodborne illness in Florida.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-based illness in the United States. Real-time PCR, pandemic group-specific PCR, ribotyping, and multilocus sequence typing were used to characterize 30 strains of V. parahaemolyticus including 11 strains associated with foodborne outbreaks in Florida and 6 known pandemic strains. Thirteen strains were positive for four pandemic group-specific PCR markers, including 5 strains associated with outbreaks in Florida. Molecular typing methods were used to further define the pandemic status of these strains because the current PCR markers are not sufficient to identify pandemic isolates. Nine of the Florida strains clustered with a majority of the known pandemic strains, based on ribotyping patterns using PvuII, but no isolated pandemic branch was formed. Using multilocus sequence typing, it was determined that 14 strains possess a previously determined pandemic sequence type. This study identified 13 novel sequence types and seven to nine novel alleles for each locus. Furthermore, the results indicate that seven of the strains from recent foodborne outbreaks in Florida are pandemic strains, and that multilocus sequence typing was the most accurate molecular method to identify these strains.

Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak.

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.