Activity of gallidermin on Staphylococcus aureus and Staphylococcus epidermidis biofilms

Abstract

Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, gallidermin's killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising "persister" cells of an unknown genetic and physiological state. Like many other antibiotics, gallidermin showed only limited activity on cells within mature biofilms.

Fig 1

Growth curves of S. aureus SA113 and S. epidermidis O47 after treatment with gallidermin at 1×, 2×, 4×, and 8× MIC of gallidermin. Gallidermin was added at the mid-exponential growth phase, after 4 h of cultivation. The MIC values of gallidermin for S. aureus SA113 and S. epidermidis O47 were 8 and 4 μg/ml, respectively.

Fig 2

Biofilm formation of S. aureus SA113 and S. epidermidis O47 in the presence of subinhibitory concentrations of gallidermin. Growth and biofilm formation occurred in microtiter plates. Shown are the turbidity (the OD₆₀₀) of the cell culture (A), the absorbance (A₅₇₀) of stained cells attached to the surface after washing (B), and photos of biofilm cells attached to microtiter wells after crystal violet staining (C). Differences were considered significant at P < 0.05.

Fig 3

Gallidermin and viability of bacterial cells in 24-h and 5-day established biofilms of S. aureus SA113 and S. epidermidis O47. The viability of bacterial cells in 24-h and 5-day biofilms was detected by MTT assay (A) or viable count (B). Differences were considered significant at P < 0.05.

Fig 4

Effects of gallidermin on atl and ica transcription. RNA from S. aureus SA113 and S. epidermidis O47 was taken from 4-, 6-, and 8-h cultures (with or without 4× MIC gallidermin treatment) and analyzed by Nothern blotting. 23S rRNA was used as a control.

Fig 5

Autolysin zymogram profiles. S. aureus SA113 and S. epidermidis O47 were treated with or without 4× MIC of gallidermin, and the cell wall-associated proteins were separated by SDS-PAGE with heat-inactivated Micrococcus luteus cells as the substrate. Proteins with autolysin activity were visualized as clear (white) bands.