Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance

Abstract

Four clinical U.S. glycopeptide intermediate resistant Staphylococcus aureus (GISA) isolates were resistant to Triton X-100-induced autolysis. Similar resistance was demonstrated in an isolate obtained after a single passage of a susceptible clinical isolate in low-level vancomycin. Strains with the vancomycin-induced Triton X-100 resistance phenotype produced active murein hydrolases but were resistant to lysis by murein hydrolases.

FIG. 1.

Triton X-100 autolysis assay of clinical GISA isolates. GISA isolates IL-F, MI, PC, and NJ are clinical isolates from Illinois, Michigan, Port Chester, N.Y., and New Jersey, respectively (6-9, 21, 23). Cultures were grown to mid-logarithmic growth phase and suspended in buffered 0.05% Triton X-100. At 1-h intervals, an aliquot was collected for absorbance determinations at 600 nm and for determination of viable cell count (CFU/ml). Absorbances are represented as percentages of the absorbance at 600 nm relative to that at time zero for each sample.

FIG. 2.

Triton X-100 autolysis assay of vancomycin-susceptible strain 523 and its vancomycin-exposed derivatives, 523a to 523k. Cultures were grown to mid-logarithmic growth phase and suspended in buffered 0.05% Triton X-100. At 1-h intervals, an aliquot was collected for determinations of absorbance at 600 nm and viable cell count (CFU/ml). Absorbances are represented as percentages of the absorbance at 600 nm relative to that at time zero for each sample.

FIG. 3.

Clustal W alignment of the Atl amino acid sequences of strains IL-A and IL-F (accession number AF537210), N315 (protein ID: BAB42150.1), RN450 (protein ID: BAA04185.1), MW2 (protein ID: AP004825.1), and 8325/4 (protein ID: AAA99982.1). Asterisks indicate positions with a single, fully conserved residue; colons indicate positions with polymorphism but with one of the following strong groups fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW; periods indicate positions with one of the following weaker groups fully conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY. Strong and weak groups are defined as a strong score of >0.5 and a weak score of ≤ 0.5, respectively, according to the Gonnet Pam250 matrix.

FIG. 4.

Zymograms containing overlays of heat-killed cells from strains IL-A and IL-F showing resistance to murein hydrolases extracted from the extracellular (EC), cell wall (CW), and intracellular (IC) fractions from strains IL-A and IL-F. Shown are murein hydrolases extracted from strains IL-A (lanes 1) and IL-F (lanes 2).

FIG. 5.

Intracellular murein hydrolases evaluated on zymograms containing overlays of heat-killed cells from M. luteus (A) or S. aureus strain 523 (B). Lanes: 1, MH from strain IL-A; 2, MH from strain IL-F; m1, Bio-Rad Kaleidoscope prestained standards; m2, Bio-Rad High Range prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard.