Alpha-toxin is required for biofilm formation by Staphylococcus aureus

Abstract

Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.

FIG. 1.

The hla mutant is defective in biofilm formation. (A) Biofilm formation phenotypes. Biofilm formation by the wild type (WT; S. aureus 8325-4) and a hla::erm mutant (S. aureus DU1090) was quantitated. Crystal violet was used to stain cells adhering to polystyrene after 8 h of growth at 37°C. (B) Complementation of the hla::erm mutant. Biofilms formed on polystyrene (8 h at 37°C) were analyzed for an hla::erm mutant harboring a vector control plasmid (pNC1) or a plasmid providing a wild-type copy of the hla gene (pDU1212 [hla⁺]).

FIG. 2.

Direct visualization of attachment phenotypes. Bacteria were inoculated onto 24-well plates, incubated for 8 h at 37°C, and then analyzed by phase-contrast microscopy. Dark areas are the adherent bacteria, and the light grayish regions represent the surface of the 24-well plate. The magnification is ×1,050. WT, wild type.

FIG. 3.

Phenotypes of cells under flow conditions. Biofilms of the wild-type (WT; S. aureus 8325-4) and the hla::erm mutant (S. aureus DU1090) strains were grown in flow cell chambers. At the times indicated, biofilms were observed from a top-down perspective by using phase-contrast microscopy. In images from 4 and 8 h, light regions represent bacterial macrocolonies (indicated by the white arrows) and dark areas are the surface of the flow cell chamber. The magnification is ×675 for images from 4 and 8 h. At 24 h, the images were recorded at an original magnification of ×230. (They are shown at a magnification of ×173.) In these images from 24 h, the very dense macrocolonies formed by the wild type appear as dark regions and the light areas define macrocolony borders or channels between the macrocolonies. Small clusters of cells, but no macrocolonies, were observed for the hla mutant at all time points.