Subinhibitory concentrations of protein synthesis-inhibiting antibiotics promote increased expression of the agr virulence regulator and production of phenol-soluble modulin cytolysins in community-associated methicillin-resistant Staphylococcus aureus

Abstract

Tetracycline, clindamycin, and other protein synthesis inhibitors at subinhibitory concentrations significantly increased the expression of the pivotal virulence regulator agr and production of the agr-regulated cytolytic phenol-soluble modulins in the community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300. Our results suggest that such protein synthesis inhibitors may exacerbate the progression of CA-MRSA disease when applied at concentrations that are too low or when treating infections caused by strains resistant to those antibiotics.

FIG. 1.

Effects of antibiotics at subinhibitory concentrations on the production of PSMs. Different antibiotics, at the concentrations shown in Table 1, were added to cultures of S. aureus strains at the time of inoculation (1:100 from precultures) into 5-ml tubes filled with 1 ml of tryptic soy broth. Tubes were shaken for 24 h at 200 rpm, and PSM concentrations were determined in the culture filtrates using RP-HPLC/ESI-MS (14). Growth under the same conditions was monitored by determining the optical density at 600 nm (OD₆₀₀). PSM concentrations in S. aureus LAC (USA300) (A), growth curves for S. aureus LAC (USA300) (B), PSM concentrations in S. aureus Sanger 252 (C), growth curves for S. aureus Sanger 252 (D). The parts of bars that are striped show the amount of N-deformylated (df) PSM among total PSM peptide concentration. Concentrations of PSMα1 are shown as an example of PSMα peptides. Statistical analysis is shown for the effects and antibiotics described in the text (Er, Te, Cl, Lin for both strains, Ox for LAC, and Mu for Sanger 252); **, P < 0.01; ***, P < 0.001; con, control (without antibiotic). Error bars represent means ± standard deviations.

FIG. 2.

Activity of agr in comparison to psmα and psmβ transcript levels under the influence of subinhibitory concentrations of tetracycline, clindamycin, and oxacillin. The activity of agr was determined by real-time quantitative reverse transcription-PCR (qRT-PCR) of RNAIII in strain LAC (USA300). The expression of psm operons was determined by qRT-PCR of psmα and psmβ transcripts under the same conditions (after 8 h of growth in shaken 125-ml flasks). Primers and conditions for qRT-PCR were as described, using the gyrB transcript as a control (13-14). Experiments were performed in triplicate. Error bars represent means ± standard deviations. The antibiotic concentrations used are shown in Table 1. **, P < 0.01; ***, P < 0.001.