Streptococcus iniae virulence is associated with a distinct genetic profile

Abstract

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10(2) CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10(7)) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10(8) CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.

FIG. 1

Weight change observed in hairless, female mice 72 h after subcutaneous injection with disease-associated (shaded bars) and commensal (open bars) strains of S. iniae. Bars represent median weight gain ± range. ∗, significantly different (P < 0.005) from weight change in the Cytodex control group as calculated by the Wilcoxon signed-rank test.

FIG. 2

Resistance to phagocytosis of S. iniae strains in whole blood. Bars indicate the percentage of viable organisms relative to initial inoculum (100%) remaining after 1.5 h of rotation in fresh human blood. The results represent the mean ± SD for disease-associated (shaded bars) and commensal (open bars) strains.

FIG. 3

Injury to BMEC monolayers exposed to disease-associated (9117) and commensal (9066) strains of S. iniae (5.0 × 10⁸ CFU) for 3 h in comparison to the cytolytic GBS strain A909 (5.0 × 10⁷ CFU). The higher inoculum used for S. iniae was required in order to obtain equivalent results. Bars indicate the mean LDH released, ± SD, relative to total LDH released from lysed monolayers.

FIG. 4

Electron micrograph of HEp-2 epithelial cells exposed to disease-associated S. iniae strain 9117 illustrating streptococci internalized within a membrane-bound vesicle.

FIG. 5

Invasion of HEp-2 epithelial cells by S. iniae disease-associated strain 9117 in the presence of the actin polymerization inhibitor cytochalasin D (CD). Bars indicate the mean level of invasion ± SD at increasing concentrations of inhibitor.

FIG. 6

BMEC adherence (shaded bars) and invasion (open bars) by disease-associated (9117) and commensal (9066) strains of S. iniae in comparison to the invasive GBS strain COH1. Bars indicate the mean adherence/invasion, ± SD, relative to the initial inoculum.