Increased glycan chain length distribution and decreased susceptibility to moenomycin in a vancomycin-resistant Staphylococcus aureus mutant

Abstract

A vancomycin-resistant Staphylococcus aureus mutant, COL-VR1 (MIC, 16 microg/ml), was isolated from methicillin-resistant S. aureus COL by exposure to vancomycin. COL-VR1 also showed decreased susceptibility to teicoplanin (8-fold), methicillin (2-fold), macarbomycin (8-fold), and moenomycin (16-fold). Macarbomycin and moenomycin are thought to directly inhibit transglycosylase activity. Characterization of the mutant revealed a thickened cell wall and suppression of penicillin-induced lysis, although the amounts of the five penicillin-binding proteins (PBPs 1, 2, 3, 4, and 2') and the profiles of peptidoglycan hydrolases were not altered. Analysis of muropeptide profile and glycan chain length distribution by reversed-phase high-pressure liquid chromatography revealed slightly decreased peptide cross-linking and an increased average glycan chain length compared to those of the parent. These results together suggest that a transglycosylase activity was enhanced in the mutant. This may represent a novel mechanism of glycopeptide resistance in S. aureus.

FIG. 1.

Population analysis of COL (closed symbols) and COL-VR1 (open symbols). S. aureus cells cultured overnight were plated on TSA containing serial dilutions of methicillin (squares) or vancomycin (circles).

FIG. 2.

Growth curves of COL (a and c) and COL-VR1 (b and d) in the presence of methicillin (c and d) or vancomycin (a and b). A small portion of S. aureus cells cultured overnight was inoculated into fresh TSB and incubated at 37°C with shaking. Antibiotics were added at the times indicated by the arrows. The OD₆₆₀ was monitored at 30-min intervals. Symbols: for panels a and b, ▪, control; •, vancomycin at 2 μg/ml; ▴, vancomycin at 4 μg/ml; ⧫, vancomycin at 8 μg/ml; □, vancomycin at 16 μg/ml; ○, vancomycin at 32 μg/ml; for panels c and d, ▪, control; •, methicillin at 10 μg/ml; ▴, methicillin at 100 μg/ml; ⧫, methicillin at 1,024 μg/ml; □, methicillin at 2,048 μg/ml.

FIG. 3.

Thin sections of S. aureus COL and COL-VR1. Exponentially growing cultures of S. aureus cells were prepared for electron microscopy as described in Materials and Methods. Bars, 100 nm.

FIG. 4.

HPLC analysis of peptidoglycan. Peptidoglycan was digested with mutanolysin (A and D) or lysostaphin (B and E), or both (C and F), and was subjected to HPLC. Data for COL are depicted in panels A, B, and C; and those for COL-VR1 are shown in panels D, E, and F.

FIG. 5.

Western blotting and zymographic analyses of S. aureus. (a) Membrane fractions of COL and COL-VR1 were analyzed by immunoblotting with serum with anti-PBP 1, anti-PBP 2, anti-PBP 3, anti-PBP 4, and anti-PBP 2′ antibodies. (b) Lysates of whole COL and COL-VR1 cells were subjected to zymography with a gel with M. luteus (b1) or a gel with S. aureus (b2). Protein marker bands are indicated at the left of the panel.