Comparison of the pathogenic potentials of environmental and clinical vibrio parahaemolyticus strains indicates a role for temperature regulation in virulence

Abstract

Although the presence of pathogenic Vibrio spp. in estuarine environments of northern New England has been known for some time (C. H. Bartley and L. W. Slanetz, Appl. Microbiol. 21: 965-966, 1971, and K. R. O'Neil, S. H. Jones, and D. J. Grimes, FEMS Microbiol. Lett. 60:163-167, 1990), their virulence and the relative threat they may pose to human health has yet to be evaluated. In this study, the virulence potential of 33 Vibrio parahaemolyticus isolates collected from the Great Bay Estuary of New Hampshire was assessed in comparison to that of clinical strains. The environmental isolates lack thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh, respectively. Though not hemolytic, they do possess putative virulence factors, such type III secretion system 1, and are highly cytotoxic to human gastrointestinal cells. The expression of known and putative virulence-associated traits, including hemolysin, protease, motility, biofilm formation, and cytotoxicity, by clinical reference isolates correlated with increased temperature from 28°C to 37°C. In contrast, the environmental isolates did not induce their putative virulence-associated traits in response to a temperature of 37°C. We further identified a significant correlation between hemolytic activity and growth phase among clinical strains, whereby hemolysin production decreases with increasing cell density. The introduction of a tdh::gfp promoter fusion into the environmental strains revealed that they regulate this virulence-associated gene appropriately in response to temperature, indicating that their existing regulatory mechanisms are primed to manage newly acquired virulence genes.

FIG. 1.

Cytotoxicities of clinical strains and select environmental isolates relative to that of reference strain BB22. (A and B) Average cytotoxicity of clinical (A) and environmental (B) strains toward human Caco-2 cells at 28°C (white bars) and 37°C (gray bars) relative to the cytotoxicity of BB22. Cytotoxicity was determined in four separate experiments; however, because of a high level of variability, the values for one representative experiment are shown (the trends were consistent for the strains shown here throughout all experiments). Values represent the averages of three replicates; error bars show standard errors. One-way ANOVAs were performed for all strains, and a significant difference relative to the result for BB22 is denoted by “a” for a significant increase in relative cytotoxicity at 28°C and “b” for a significant decrease at 37°C; “c” denotes a statistically significant change in a single strain at 37°C compared to its cytotoxicity at 28°C.

FIG. 2.

Quantitative hemolysis by a select clinical strain at 37°C compared to cell density. Red blood cell lysis was determined over time, and relative hemolytic activity was determined (OD₄₁₅/OD₆₀₀) for one representative strain, MDOH-03-17282-Vp (bars), throughout the growth cycle (black line; OD₆₀₀).

FIG. 3.

Induction of the tdh promoter in clinical and environmental strains. Promoter activity of a tdh::gfp promoter fusion harbored in trans in a low-copy-number plasmid is presented as relative fluorescence (fluorescence/OD₆₀₀) for various strains during maximal production (OD₆₀₀ ∼ 1.0) as determined from time course assays for cultures grown at 37°C.