Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans

Abstract

Background: Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor.

Methods and findings: We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB(-) mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 +/- 1 d versus 7 +/- 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not.

Conclusions: The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.

Figure 1. Study Design

First, the left or right nostril was randomized to receive the inoculation mix, containing strains 8325-4 (WT) and DU5997 (mutant strain). Second, the contralateral nostril was randomized to receive either the naturally occurring (“WT” in figure) or mutant strain.

Figure 2. ClfB Expression Experiments

(A) Bacterial growth and ClfB expression in RPMI. Strains 8325–4, DU5997, and SH1000 were grown in RPMI, and samples were taken at regular intervals to monitor growth. (B) Samples taken at early exponential, early stationary, and late stationary phases of growth were analyzed by Western immunoblotting. Lanes 1, 2, and 3 show ClfB expression from strain 8325–4 at early exponential, early stationary, and late stationary growth phases, respectively. Lanes 4, 5, and 6 show ClfB expression from strain DU5997 at early exponential, early stationary, and late stationary growth phases, respectively. Lanes 7, 8, and 9 show ClfB expression from SH1000 at early exponential, early stationary, and late stationary growth phases, respectively. (C) Bacterial growth and ClfB expression in TSB. Strain 8325–4 was grown in TSB, and samples were taken at regular intervals to monitor growth. (D) Western immunoblotting was carried out to detect ClfB expression at early exponential, early stationary, and late stationary phases of growth. Lanes 1, 2, and 3 show ClfB expression from strain 8325–4 grown in TSB at early exponential, early stationary, and late stationary growth phases, respectively.

Figure 3. S. aureus Adherence to Recombinant Cytokeratin 10 in Different Growth Stages

Data are epresentative of three independent experiments. Bars represent standard deviation. (A) Adherence of bacterial cells in exponential phase of growth to rMK10. Strain 8325–4 was grown to exponential phase in RPMI or TSB, and its ability to bind rMK10 was compared to DU5997 grown in RPMI. (B) Adherence of bacterial cells in stationary phase of growth to rMK10. Strain 8325–4 was grown to stationary phase in RPMI or TSB, and its ability to bind rMK10 was compared to DU5997 grown in RPMI.

Figure 4. Survival in the Nose of the Wild-Type Strain versus the Mutant Strain

(A) Kaplan-Meier survival curve of the 8325–4 S. aureus (solid line) and its ClfB⁻ mutant (DU5997) strain (dashed line) in human nose (last cultured strain in either nostril). All cells of the mutant strain were eliminated after a period of 14 d, which is significantly faster than the elimination rate for the 8325–4 S. aureus strain. (B) Kaplan-Meier survival curve of the 8325–4 S. aureus (WT) and ClfB⁻ mutant (DU5997; dashed line) that were inoculated together as a mix in one nostril. (C) Number of cfu of 8325–4 and DU5997 in follow-up samples.