Very low ethanol concentrations affect the viability and growth recovery in post-stationary-phase Staphylococcus aureus populations

Abstract

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (< or =0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC(+) conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC(-) conditions) for prolonged culture times (up to 8 days). VLEC(+)-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC(+) conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC(+) conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.

FIG. 1.

Effects of VLEC and arginine on micromorphology of S. aureus DSM20231. Shown are representative scanning electron micrographs of S. aureus DSM20231 grown for various times (24 h, 48 h, 72 h, 120 h, and 192 h) in unsupplemented medium (A to E, top to bottom), under VLEC⁺ conditions (F to J, top to bottom), or under VLEC⁺ conditions and supplemented with 5 mM arginine (K to O, top to bottom).

FIG. 2.

Analysis of long-term growth, stationary-phase survival, and membrane potential of S. aureus. (A) Growth analysis (OD₆₀₀) of S. aureus DSM20231 under VLEC⁻ (▪) and VLEC⁺ (•) conditions, determined in BHI medium. Single colonies were inoculated into BHI in the absence (▪) or presence (•) of 0.1% (vol/vol) ethanol and incubated at 37°C under microaerophilic conditions for up to 8 days. Data are means ± standard errors of the means of values obtained in three independent experiments. *, P < 0.05; **, P < 0.001 (t test). (B) Viability of S. aureus DSM20231. After growth for the indicated time under VLEC⁻ (▪) or VLEC⁺ (•) conditions, aliquots were removed and CFU/ml was determined in triplicate. Data are means ± standard deviations of values obtained in two independent experiments. *, P < 0.05; **, P < 0.005 (t test). (C) Ethanol concentration-dependent delayed recovery. (D) Membrane potential measurement of S. aureus DSM20231. Addition of ethanol (0.1%) is indicated by an arrow. Data are representative of two independent experiments.

FIG. 3.

Analysis of ethanol in the culture supernatant and effect of alcohol-aldehyde dehydrogenase (adhE) in VLEC exposure. (A) Determination of ethanol levels in the culture supernatant of S. aureus DSM20231 under VLEC⁻ (▪) and VLEC⁺ conditions (•) at the indicated time points. (B) Estimation of the nonenzymatic loss of ethanol from the culture supernatant with (▪) or without (•) bacteria under VLEC⁺ conditions using gas chromatography. (C) Real-time RT-PCR quantification of alcohol-aldehyde dehydrogenase (adhE) gene expression in S. aureus with or without VLEC at different time points. Expression of the adhE gene in S. aureus DSM20231 was determined in populations grown under VLEC⁻ or VLEC⁺ conditions by real-time RT-PCR at different times as described in Materials and Methods. Shown are transcript quantities relative to the internal control (gyrB) transcript, expressed as n-fold increase. The x axis denotes time (hours). Data are representative of two independent experiments.

FIG. 4.

Analysis of external pH and levels of metabolites of the culture supernatant. External pH (A) and levels of glucose (B), acetate (C), and ammonia (D) in the culture supernatant of S. aureus DSM20231 were determined under VLEC⁻ (▪) and VLEC⁺ (•) conditions at the indicated time points. Data are representative of two independent experiments.

FIG. 5.

Depletion of free amino acids from the BHI medium. Shown are concentrations of free amino acids l-serine (A), l-glycine (B), l-arginine (C), l-glutamic acid (D), l-ornithine (E), and l-proline (F) in BHI culture medium of S. aureus DSM20231 grown under VLEC⁻ (□) and VLEC⁺ (○) conditions. Data are mean molar concentrations (nmol/ml) ± standard deviations of two independent experiments.

FIG. 6.

(A) Effect of l-arginine supplementation. S. aureus DSM20231 was grown under VLEC⁻ (▪) or VLEC⁺ (•) conditions, or in VLEC supplemented with 2 mM l-arginine (▴), or in VLEC with 5 mM l-arginine (▾) in BHI medium, and cell densities were determined as described in Fig. 2. Data are representative of two independent experiments. (B) Real-time RT-PCR quantification of microaerobic arcA (arginine deiminase) gene expression. Expression of the arcA gene in S. aureus DSM20231 populations grown with or without VLEC was determined by real-time RT-PCR at different time intervals as described in Materials and Methods. Shown are transcript quantities relative to the internal control (gyrB) transcript, expressed as n-fold increase.