Correlation of daptomycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid production and D-alanylation

Abstract

Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycin-resistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycin-resistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.

Fig. 1.

TEM analyses of the daptomycin-susceptible parental strain (616), the vancomycin-exposed but daptomycin-unexposed strain (621), and the two daptomycin-resistant strains (701 and 703). The thickness of cell walls (in nanometers) was measured at ×190,000 magnification. Data (± SD) for strains 616, 701, and 703 have been recently published (37).

Fig. 2.

Muropeptide pattern. The cell wall of all four strains was isolated and digested by muraminidase mutanolysin and analyzed by HPLC. The muropeptide patterns of strains 616, 701 and 703 were always very similar. Differences in the muropeptide pattern of strain 621 compared to those of the other three strains are indicated by black arrows.

Fig. 3.

Determination of O-acetylated muropeptides by HPLC analysis. The cell walls of all three strains were isolated and enzymatically digested into monomeric muropeptides by mutanolysin and lysostaphin. The O-acetylated and nonacetylated muropeptides were separated by HPLC, and the content of the two most prevalent peak groups was determined by MS. The peak group at retention time 60 to 75 min contains nonacetylated muropeptides, while the muropeptides at the peak retention times of 75 to 85 min are O-acetylated.

Fig. 4.

Analysis of the amount of the peptidoglycan precursor UDP-MurNAc-pentapeptide. The peptidoglycan precursor UDP-MurNAc-pentapeptide was isolated from growing cells, and its amount for each strain was detected by HPLC (blue, 616; red, 701; green, 703).

Fig. 5.

Relative transcript levels of the dltA gene and tagA gene in strains 616 and 701. Values from exponential and stationary growth phase RNA samples were normalized versus housekeeping gene, gyrB, expression levels; data from strain 616 were set to 1 to allow comparison of data from different samples. * P < 0.05 and ** P < 0.001 versus 616.