Evaluation of a tissue culture-based approach for differentiating between virulent and avirulent Vibrio parahaemolyticus strains based on cytotoxicity.

The ability of only a subset of Vibrio parahaemolyticus strains to cause human infection underscores the need for an analytical method that can effectively differentiate between pathogenic strains and those that do not cause disease. We tested the feasibility of a tissue culture-based assay to determine whether clinical isolates could be differentiated from nonclinical isolates based on relative isolate cytopathogenicity. To screen for cytotoxic capability, we measured relative extracellular lactate dehydrogenase as an indicator of host cell damage in five different mammalian cell lines in the presence of V. parahaemolyticus. Isolates originating from clinical sources exhibited 15.5 to 59.3% relative cytotoxicity, whereas those originating from food sources exhibited 4.4 to 54.9% relative cytotoxicity. In the presence of -1.2 x 10(6) cells, cytotoxicity was 1.6- to 3.5-fold higher (P < 0.05) for clinical isolates than for nonclinical isolates in L2, Henle 407, and Caco-2 cell lines. V. parahaemolyticus serotype O3:K6 clinical isolates had 1.6- to 2.1-fold higher cytotoxicity than did the non-O3:K6 clinical isolates, with significantly higher cytotoxicity in HeLa, J774A.1, and Henle 407 cells than in L2 and Caco-2 cells. Because V. parahaemolyticus often is found in oysters, the effect of the presence of an oyster matrix on assay efficacy was also tested with L2 cells. The cytotoxicity elicited by a highly cytotoxic V. parahaemolyticus isolate was not affected by the presence of oyster tissue, suggesting that an oyster matrix will not interfere with assay sensitivity. In the present format, this assay can detect the presence of > 10(5) cells of a virulent V. parahaemolyticus strain in an oyster matrix.

Vibrio parahaemolyticus growth under low-iron conditions and survival under high-magnesium conditions.

Since 1996, Vibrio parahaemolyticus serotype O3:K6 and closely related strains have been associated with an increased incidence of V. parahaemolyticus gastroenteritis worldwide, suggesting the emergence of strains with enhanced abilities to cause disease. One hypothesis for the recent emergence of V. parahaemolyticus O3:K6 and related strains is an enhanced capacity for environmental survival relative to other strains, which might result in increased human exposure to these organisms. Therefore, the objective of this study was to test the hypothesis that survival or growth characteristics of clinical V. parahaemolyticus isolates differ from those of nonclinical isolates under different environmental conditions. Twenty-six V. parahaemolyticus isolates selected to represent either clinical or food sources were monitored for either survival following exposure to high magnesium (300 mM) or growth under iron-limited conditions. Isolates in each category (clinical or food) differed widely in survival capabilities following 24 h of exposure to 300 mM Mg2+. Although 4 of 15 clinical isolates grew better at approximately 0.96 microM Fe2+ (iron-limited conditions) than at 50 microM Fe2+ (iron-rich conditions), as an entire group clinical isolates in this study were not more effective at growing under iron-limited conditions than were strains not associated with disease. Within the diverse collection of strains examined in these experiments, neither growth characteristics in low-iron environments nor survival capabilities following exposure to high magnesium concentrations were uniformly different between clinical and nonclinical V. parahaemolyticus isolates. Therefore, neither phenotypic characteristic can be used to reliably differentiate potentially pathogenic V. parahaemolyticus strains.

Effects of acid stress on Vibrio parahaemolyticus survival and cytotoxicity.

For several foodborne bacterial pathogens, an acid tolerance response appears to be an important strategy for counteracting acid stress imposed either during food processing or by the human host. The acid tolerance response enhances bacterial survival of lethal acid challenge following prior exposure to sublethal acidic conditions. Previous studies have revealed relationships between a foodborne pathogen's ability to survive acid challenge and its infectious dose. Vibrio parahaemolyticus is capable of causing gastroenteritis when sufficient cells of pathogenic strains are consumed. This study was designed to characterize acid sensitivities and to compare the effects of sublethal acid exposure (adaptation) on survival capabilities and cytotoxicities of different V. parahaemolyticus strains. Survival of acid challenge by stationary-phase cells differed by up to 3 log CFU/ml among the 25 isolates tested. No differences in acid resistance were found between strains when they were grouped by source (clinical isolates versus those obtained from food). Survival at pH 3.6 for log-phase cells that had been previously exposed to sublethal acidic conditions (pH 5.5) was enhanced compared with that for cells not previously exposed to pH 5.5. However, for stationary-phase cells, exposure to pH 5.5 impaired both subsequent survival at pH 3.6 and cytotoxicity to human epithelial cells. Relative cytotoxicities of nonadapted stationary-phase cells were 1.2- to 4.8-fold higher than those of adapted cells. Sublethal acid exposure appears to impose measurable growth phase-dependent effects on subsequent lethal acid challenge survival and cytotoxicity of V. parahaemolyticus.

Compartmentalization of the broad-range phospholipase C activity to the spreading vacuole is critical for Listeria monocytogenes virulence.

Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBDelta pro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBDelta pro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBDelta pro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBDelta pro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

The metalloprotease of Listeria monocytogenes controls cell wall translocation of the broad-range phospholipase C.

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.

Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections.

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.

Comparative phenotypic, molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates.

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.