In Vitro Time-kill Activities of Rifalazil, Alone and in Combination with Vancomycin, against Logarithmic and Stationary Cultures of Staphylococcus aureus

Abstract

Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the b subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that 1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, 2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log 10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, 3) rifalazil and vancomycin in combination killed stationary phase cultures by 3ϳ4 Log 10 by 48 hours.

Enhanced Activity of Rifalazil in Combination with Levofloxacin, Linezolid, or Mupirocin against Staphylococcus aureus In Vitro

“…Bactericidal activity was assessed over 24 hours using agar plate counts to determine CFU. The high initial density allowed us to monitor the appearance of rifamycin-resistant mutants, as described previously [3]. We found that for log phase (growing) cells, when compared with each drug used alone, rifalazil co-treatment with levofloxacin, linezolid, or mupirocin showed enhanced killing of S. aureus and suppression of rifamycin-resistant mutants.…”

“…In this set of experiments, cultures were followed for at least 24 hours, and those that showed any trend toward decreased viability at 24 hours were followed for an additional 24 hours. Rifalazil plus vancomycin, previously shown to have activity against stationary phase cells [3] and therefore used as a positive control, again showed effective bactericidal activity. Our data indicate that the rifalazil-vancomycin combination appears to be thus far uniquely effective against non-growing S. aureus.…”

“…aureus strain ATCC 29213, which was used in this study, is a good representative for S. aureus species because the MIC of rifalazil for this strain is somewhat higher than the MIC 50 for MSSA and MRSA clinical isolates [14]. Timekill curves were carried out as described previously for high density cultures [3]. Briefly, 3ϳ5 colonies of S. aureus strain ATCC 29213, grown on a Mueller-Hinton agar (Becton Dickinson) plate at 35°C for 18 hours, were inoculated into 50ϳ100 ml of cation-adjusted MuellerHinton broth in a 500 ml flask and grown at 37°C with shaking.…”

“…We investigated previously whether rifalazil could be used in combination with vancomycin to produce both potent bactericidal activity and the suppression of resistant mutants, and found that co-treatment of either log or stationary phase Staphylococcus aureus cells with rifalazil and vancomycin increased the bactericidal activity over that obtained using either drug alone and delayed the appearance of resistant mutants [3]. To continue our study of strategies to address drug resistance, we assessed the effects of rifalazil co-treatment with the antibiotics linezolid, mupirocin, or levofloxacin on S. aureus by means of in vitro time kill curves, as previously described [3].…”

“…To continue our study of strategies to address drug resistance, we assessed the effects of rifalazil co-treatment with the antibiotics linezolid, mupirocin, or levofloxacin on S. aureus by means of in vitro time kill curves, as previously described [3].…”