Stepwise decrease in daptomycin susceptibility in clinical Staphylococcus aureus isolates associated with an initial mutation in rpoB and a compensatory inactivation of the clpX gene

Abstract

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.

FIG 1

Population analysis profiles. Overnight cultures of SADR-1, SADR-2, SADR-3, and SADR-4 were serially diluted and plated on agar plates with increasing concentrations of daptomycin (with 50 mM Ca²⁺) (A), vancomycin (in this experiment, the hVISA strain MU3 was included as a control) (B) or oxacillin (C), as indicated. Representative data from three individual experiments are shown.

FIG 2

Reduced cell size and increased cell wall thickness in SADR-2 and SADR-3. Representative TEM images of SADR-1, SADR-2, SADR-3, and SADR-4 are shown. The table shows the results of the quantitative analysis of cell diameter and cell wall thickness for the four strains. The average cell diameters of SADR-2, SADR-3, and SADR-4 were significantly different from the average cell diameter of SADR-1 (P < 0.001), and the cell wall thicknesses of SADR-2 and SADR-3 were significantly different from the cell wall thickness of SADR-1 (P < 0.001). The cell wall thickness of SADR-4 was significantly different from that of SADR-1 (P = 0.005), as shown using the unequal variances Student's t test.

FIG 3

The absence of ClpX improves growth and survival at high temperatures. (A) Western blot detection of ClpX in total protein extracts derived from cells in late exponential growth phase confirms that ClpX is not produced in SADR-3 and SADR-4; (B) cultures of SADR-1 (parental isolate), SADR-2, SADR-3, and SADR-4 were grown to exponential phase in TSB at 37°C. At an OD₆₀₀ of 0.5, the cultures were diluted 10⁻¹-, 10⁻²-, 10⁻³-, and 10⁻⁴-fold, and 10 μl of each dilution was spotted onto TSA plates that were incubated at the indicated temperatures.

FIG 4

The Spx-controlled stress regulon is upregulated in SADR-2, SADR-3, and SADR-4. (A) Western blot detection of Spx. Cellular proteins were extracted from an equal number of cells in late exponential phase (OD₆₀₀ = 1.0 ± 0.1). The extracted proteins were separated by SDS-PAGE, blotted onto a PVDF membrane, and probed with anti-Spx antibody. (B) Northern blot detection of the trxB transcript. RNA was isolated from cells in exponential growth phase (OD₆₀₀ = 0.7) and in postexponential growth phase (OD₆₀₀ = 2.0 ± 0.1). Transcripts were identified using a radioactively labeled trxB-specific probe. To visualize equal loading, a picture of the ethidium bromide-stained agarose gel is shown in the bottom panel. The numbers at the bottom indicate the amount of trxB mRNA relative to the signal obtained in exponential-phase cells of SADR-1 (which was given a value of 1.0) using a Cyclone Plus phosphorimager from PerkinElmer.

FIG 5

The genetic changes selected after daptomycin treatment reduce the expression of virulence factors. (Top) Western blot detection of protein A. Cellular proteins were extracted from an equal number of cells in late exponential phase (OD₆₀₀ = 1.0 ± 0.1). (Middle) Hemolytic activity detected as clearing zones. Overnight cultures of SADR-1, SADR-2, SADR-3, and SADR-4 were streaked on plates containing 5% calf blood. (Bottom) Extracellular proteolytic activity was examined on plates containing 10% skimmed milk. Clear zones are indicative of casein degradation.