Enhanced post-stationary-phase survival of a clinical thymidine-dependent small-colony variant of Staphylococcus aureus results from lack of a functional tricarboxylic acid cycle

Abstract

The mechanisms underlying the persistence of the Staphylococcus aureus small-colony variant (SCV) are not fully elucidated. In this study, clinical thymidine-dependent SCVs displayed altered expression of citB, clpC, and arcA genes, reduced acetate catabolization, and enhanced survival. These results implicate the importance of changes in tricarboxylic acid cycle and acetic acid metabolism in SCV survival and persistence.

FIG. 1.

(A) Long-term growth analysis of normal (non-TD) S. aureus and TD-SCV. Growth curves (optical density at 600 nm [OD₆₀₀]) of normal S. aureus (▪) and TD-SCV (•) were determined in BHI medium under aerobic conditions. Arrows indicate the time points for total RNA extraction. The data shown are means ± standard errors of the means (error bars) for values obtained from three independent experiments. Values that are significantly different from the values for normal S. aureus (P < 0.001 by t test) are indicated (*). (B) Thymidine limitation in TD-SCV S. aureus enhances post-stationary-phase survival. Cultural aliquots were removed at different time intervals of normal S. aureus (▪) and TD-SCV (•) from the growth analysis cultures, plated on BHI agar plates, and incubated at 37°C for 24 h to 48 h. The values (CFU per milliliter) were determined in triplicate. The data shown are means ± standard deviations (error bars) for values obtained from two independent experiments. The indicated increases (0.1-fold and 4.1-fold increases) relate CFU counts of the TD-SCV with those of normal S. aureus at respective time points (4 h and 120 h) and refer to differences in viability of the TD-SCV versus normal S. aureus (please refer also to the calculated “survival advantage” in “Thymidine auxotrophism impairs exponential-phase growth but enhances stationary-phase survival in the TD-SCV.”). The comparison of the two curves as a whole shows that the curves are significantly different referring to the population as well as the incubation time (two-factor analysis of variance, P ≤ 0.001), with significant differences at select time points (*, P ≤ 0.05 compared to normal S. aureus [t test]).

FIG. 2.

Determination of external pH, glucose concentration, and levels of metabolites (ammonia, acetate, and lactate) in the culture supernatant. At different time intervals, supernatants of normal (non-TD) S. aureus (▪) and TD-SCV (•), cultivated in BHI medium, were analyzed for (A) external pH and (B) ammonia, (C) glucose, (D) acetate, and (E) lactate concentrations. External pH data are means ± standard deviations (error bars) for values obtained from three independent experiments. Values that are significantly different from the values for normal S. aureus (t test) are indicated (*, P < 0.01; **, P < 0.001). Ammonia, glucose, acetate, and lactate data are representative results of at least two independent experiments.

FIG. 3.

Real-time reverse transcription-PCR quantification. Gene expression of (A) citB (encoding aconitase), (B) clpC (encoding ClpC ATPase) and (C) arcA (encoding arginine deiminase) was determined in normal (non-TD) S. aureus and TD-SCV at exponential growth phase (EP) and stationary growth phase (SP) by real-time reverse transcription-PCR. The transcript quantities relative to the values for an internal control (gyrB) transcript expressed as changes in the increase (n-fold increase) are shown. Data are means plus standard errors of the means (error bars) for values obtained from three independent experiments. (EP for normal S. aureus is 2.5 h, while SP is 7 h; EP for TD-SCV is 4.5 h, while SP is 9 h).