Gram-negative bacteria remain clinically important pathogens in both hospital and community settings. Recent research indicates that efflux pumps play a prominent role in the multidrug resistance of Pseudomonas aeruginosa and many other gram-negative bacteria. Four multidrug efflux pump systems have been well characterized in P. aeruginosa: MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM. These efflux pumps have different substrate specificities, and their production and activity can be increased by many factors commonly present in infections (e.g., high inocula of bacteria, low pH, and stationary-phase growth). Moreover, fluoroquinolone antibiotics can commonly select mutants that constitutively overproduce Mex-Opr efflux pump systems. Based on most recent studies, the prevalence of efflux pump overproduction in clinical strains of P. aeruginosa may range from 14-75%. The best treatment for infections caused by bacteria that overproduce efflux pumps is unknown, but pharmacodynamic optimization of antibiotics and the use of antibiotic combinations that are substrates for different pump systems may represent reasonable strategies until more data are available.

NorA is a membrane-associated multidrug efflux protein that can decrease susceptibility to fluoroquinolones in Staphylococcus aureus. To determine the effect of NorA inhibition on the pharmacodynamics of fluoroquinolones, we evaluated the activities of levofloxacin, ciprofloxacin, and norfloxacin with and without various NorA inhibitors against three genetically related strains of S. aureus (SA 1199, the wild-type; SA 1199B, a NorA hyperproducer with a grlA mutation; and SA 1199-3, a strain that inducibly hyperproduces NorA) using susceptibility testing, time-kill curves, and postantibiotic effect (PAE) methods. Levofloxacin had the most potent activity against all three strains and was minimally affected by addition of NorA inhibitors. In contrast, reserpine, omeprazole, and lansoprazole produced 4-fold decreases in ciprofloxacin and norfloxacin MICs and MBCs for SA 1199 and 4-to 16-fold decreases for both SA 1199B and SA 1199-3. In time-kill experiments reserpine, omeprazole, or lansoprazole increased levofloxacin activity against SA 1199-3 alone by 2 log 10 CFU/ml and increased norfloxacin and ciprofloxacin activities against all three strains by 0.5 to 4 log 10 CFU/ml. Reserpine and omeprazole increased norfloxacin PAEs on SA 1199, SA 1199B, and SA 1199-3 from 0.9, 0.6, and 0.2 h to 2.5 to 4.5, 1.1 to 1.3, and 0.4 to 1.1 h, respectively; similar effects were observed with ciprofloxacin. Reserpine and omeprazole increased the levofloxacin PAE only on SA 1199B (from 1.6 to 5.0 and 3.1 h, respectively). In conclusion, the NorA inhibitors dramatically improved the activities of the more hydrophilic fluoroquinolones (norfloxacin and ciprofloxacin). These compounds may restore the activities of these fluoroquinolones against resistant strains of S. aureus or may potentially enhance their activities against sensitive strains.

The activities of the oxazolidinone antibacterial agents eperezolid (PNU-100592) and linezolid (PNU-100766) were compared with that of vancomycin against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus (n = 200), coagulase-negative staphylococci (n = 100), and vancomycin-susceptible and -resistant Enterococcus faecalisand Enterococcus faecium (n = 50). Eperezolid and linezolid demonstrated good in vitro inhibitory activity, regardless of methicillin susceptibility for staphylococci (MIC at which 90% of the isolates are inhibited [MIC90] range, 1 to 4 μg/ml) or vancomycin susceptibility for enterococci (MIC90 range, 1 to 4 μg/ml). In time-kill studies, eperezolid and linezolid were bacteriostatic in action. A postantibiotic effect of 0.8 ± 0.5 h was demonstrated for both eperezolid and linezolid against S. aureus, S. epidermidis, E. faecalis, and E. faecium.

Summary Antibiotic resistance is a rapidly evolving health concern that requires a sustained effort to understand mechanisms of resistance and develop new agents that overcome those mechanisms. The dihydrofolate reductase (DHFR) inhibitor, trimethoprim (TMP), remains one of the most important orally administered antibiotics. However, resistance through chromosomal mutations and mobile, plasmid-encoded insensitive DHFRs threatens the continued use of this agent. We are pursuing the development of new propargyl-linked antifolate (PLA) DHFR inhibitors designed to evade these mechanisms. While analyzing contemporary TMP-resistant clinical isolates of methicillin-resistant and sensitive Staphylococcus aureus, we discovered two mobile resistance elements, dfrG and dfrK. This is the first identification of these resistance mechanisms in the United States. These resistant organisms were isolated from a variety of infection sites, show clonal diversity and each contain distinct resistance genotypes for common antibiotics. Several PLAs showed significant activity against these resistant strains by direct inhibition of the TMP resistance elements.