Since its discovery about 50 years ago, Vibrio parahaemolyticus has been implicated as a major cause of foodborne illness around the globe. V. parahaemolyticus is a natural inhabitant of marine waters. Human infections are most commonly associated with the consumption of raw, undercooked or contaminated shellfish. A few individual V. parahaemolyticus virulence factors, including the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), have been investigated in depth, yet a comprehensive understanding of this organism's ability to cause disease remains unclear. Since 1996, serotype O3:K6 strains have been associated with an increased incidence of gastroenteritis in India and in Southeast Asia, and with large-scale foodborne outbreaks in the United States (US). In light of the emerging status of pathogenic V. parahaemolyticus, the US Food and Drug Administration conducted a microbial risk assessment to characterize the risk of contracting V. parahaemolyticus infections from consuming raw oysters. This review summarizes epidemiological findings, discusses recognized and putative V. parahaemolyticus virulence factors and pathogenicity mechanisms, and describes strategies for preventing V. parahaemolyticus infections.

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in com mercial products. Species identification of a bank of com mercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation anal ysis, and cellular fatty acid methyl ester analysis. Re sults from partial 16S rDNA sequencing indicated dis crepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacil lus acidophilus and Lactobacillus casei groups. Carbohy drate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probi otic lactobacilli.

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.Vibrio parahaemolyticus is reported as an agent of foodborne illness in the United States (13) and around the globe. Human infection with this pathogen is associated most frequently with the consumption of seafood, primarily raw or improperly cooked shellfish (4). Consumption of sufficiently high numbers of organisms of virulent V. parahaemolyticus strains can cause gastroenteritis, septicemia, and even death. Since 1996, an increased incidence of gastroenteritis in many parts of the world (7, 19) has been associated with V. parahaemolyticus serotype O3:K6. The recent predominance of the O3:K6 serotype as a causative agent of multiple gastroenteritis outbreaks is noteworthy, because, in general, V. parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. The association of this "new" O3:K6 serotype with large-scale food-borne disease outbreaks suggests this organism may have an unusual capacity to be transmitted by foods and/or to cause human infection.The current standard method for definitive identification of V. parahaemolyticus O3:K6 requires serotyping (9). This method is expensive, tedious, and time-consuming. Because O3:K6 isolates recovered since 1996 have been shown to be genetically distinct from previously isolated O3:K6 strains (3, 17, 19),...

Aims: The objective of this study was to generate strain-specific genomic patterns of a bank of 67 commercial and reference probiotic strains, with a focus on probiotic lactobacilli. Methods and Results: Pulsed-field gel electrophoresis (PFGE) was used as the primary method for strain differentiation. This method was compared with carbohydrate fermentation analysis. To supplement visual comparison, PFGE patterns were analysed quantitatively by cluster analysis using unweighted pair group method with arithmetic averages. SmaI, NotI and XbaI were found to effectively generate clear and easy-to-interpret PFGE patterns of a range of probiotic strains. Some probiotic strains from different sources shared highly similar PFGE patterns. Conclusions: Results document the value of genotypic strain identification methods, combined with phenotypic methods, for determining probiotic strain identity and relatedness. No correlation was found between relatedness determined by carbohydrate fermentation profiles alone compared with PFGE analysis alone. Some commercial strains are probably derived from similar sources. Significance and Impact of the Study: This approach is valuable to the probiotic industry to develop commercial strain identification patterns, to provide quality control of strain manufacturing production runs, to track use of protected strains and to determine the relatedness among different research and commercial probiotic strains.