Role of iron in human serum resistance of the clinical and environmental Vibrio vulnificus genotypes

Abstract

We recently reported a simple PCR procedure that targets a sequence variation of the virulence-correlated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C- and E-type strains of V. vulnificus and may explain why some strains (C- and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.

FIG. 1.

Survival of clinical (solid bars) and environmental (open bars) genotypes of V. vulnificus exposed to normal human serum (A) or complement-inactivated human serum (B). Each bar represents the average survival in CFU for the five strains tested of each genotype. Control columns represent cells grown in HI prior to serum exposure. Asterisks denote significant differences between genotypes at each time interval based on results of two-way ANOVA with Bonferroni posttests (*, P < 0.05; **, P < 0.01).

FIG. 2.

Serum survival of clinical (solid bars) and environmental (open bars) genotypes of V. vulnificus exposed to normal human serum for 1 h with or without additions of ferric ammonium citrate. Each bar represents the average survival in CFU for the five strains tested of each genotype. Columns marked “Initial Inocula” represent cells grown in HI prior to serum exposure. Asterisks denote significant differences in survivability between genotypes based on results of two-way ANOVA with Bonferroni posttests (***, P < 0.001).

FIG. 3.

Serum survival of viuB⁺ (▪) and viuB-negative (□) E- genotype strains of V. vulnificus exposed to normal human serum for 1 h with or without addition of ferric ammonium citrate. Each bar represents the average survival in CFU for each of the viuB⁺ or viuB-negative E-genotype strains tested. Initial inoculum columns represent cells grown in HI prior to serum exposure. Asterisks denote significant differences in survivability between genotypes based on results of two-way ANOVA with Bonferroni posttests (***, P < 0.001).

FIG. 4.

Percent survival of C-genotype (solid bars) and E-genotype (open bars) strains of V. vulnificus incubated in artificial estuarine water and then removed and exposed to human serum for 1 h. Each bar represents the average survival in CFU for the five strains tested of each genotype. Results are relative to initial inoculum (control), which represent cells grown in modified HI (1,464 mosM) prior to microcosm inoculation and serum exposure. Asterisks denote significance based on results of two-way ANOVA with Bonferroni posttests (**, P < 0.01; ***, P < 0.001).