Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis.

The cytotoxic alpha-toxin (encoded by hla) of Staphylococcus aureus is regulated by three loci, agr, sarA and sae, in vitro. Here, we assess the regulation of hla in a guinea pig model of device-related infection by quantifying RNAIII (the effector molecule of agr) and hla directly in exudates accumulating in infected devices without subculturing of the bacteria. LightCycler reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the transcripts. Strains RN6390 and Newman expressed considerably smaller amounts of RNAIII in the guinea pig than during in vitro growth. The residual RNAIII expression decreased during the course of infection and was negatively correlated with bacterial densities. As with RNAIII, the highest hla expression was detected in both strains early in infection. Even in strain Newman, a weak hla producer in vitro, a pronounced expression of hla was observed during infection. Likewise, four S. aureus isolates from cystic fibrosis (CF) patients expressed Q1hla despite an inactive agr during device-related infection as in the CF lung. Mutation of agr and sarA in strain Newman and RN6390 had no consequence for hla expression in vivo. In contrast, the mutation in sae resulted in severe downregulation of hla in vitro as well as in vivo. In conclusion, S. aureus seems to be provided with regulatory circuits different from those characterized in vitro to ensure alpha-toxin synthesis during infections.

Effects of amoxicillin, gentamicin, and moxifloxacin on the hemolytic activity of Staphylococcus aureus in vitro and in vivo.

In Staphylococcus aureus infection hemolysis caused by the extracellular protein alpha-toxin encoded by hla is thought to contribute significantly to its multifactorial virulence. In vitro, subinhibitory concentrations of beta-lactam antibiotics and fluoroquinolones increase the levels of hla and alpha-toxin expression, whereas aminoglycosides decrease the levels of hla and alpha-toxin expression. In the present study we investigated the effects of subinhibitory concentrations of amoxicillin, gentamicin, and moxifloxacin on hla and alpha-toxin expression and total hemolysis of S. aureus strain 8325-4, a high-level alpha-toxin producer, and its alpha-toxin-negative mutant, DU 1090, in vitro and in a rat model of chronic S. aureus infection. The levels of expression of hla and alpha-toxin and total hemolysis did not differ significantly when amoxicillin, gentamicin, or moxifloxacin was added to cultures of S. aureus strain 8325-4. In vivo, strain 8325-4 induced a significantly increased level of hemolysis in infected pouches compared to that in uninfected control pouches, but the hemolysis was reduced to control levels by treatment with doses of amoxicillin, gentamicin, or moxifloxacin that reduced bacterial numbers by 2 orders of magnitude. Additionally, the effects of subinhibitory concentrations of the three antibiotics on total hemolysis of four methicillin-resistant S. aureus and three methicillin-sensitive S. aureus (MSSA) clinical isolates were assessed in vitro. A significant increase in total hemolysis was observed for only one MSSA strain when it was treated with amoxicillin but not when it was treated with moxifloxacin or gentamicin. When purified alpha-toxin was incubated with purified human neutrophil elastase, alpha-toxin was cleaved nearly completely. The results suggest that the penicillin-induced increases in S. aureus alpha-toxin expression are strain dependent, that reduction of bacterial numbers in vivo counteracts this phenomenon effectively, and finally, that in localized S. aureus infections alpha-toxin activity is controlled by neutrophil elastase.