The lipopeptide daptomycin has been approved for use in skin and skin-structure infections but has failed to meet statistical noninferiority criteria in a clinical trial for severe community-acquired pneumonia. Daptomycin exhibited an unusual pattern of activity in pulmonary animal models: efficacy in Staphylococcus aureus hematogenous pneumonia and inhalation anthrax but no activity against Streptococcus pneumoniae in simple bronchial-alveolar pneumonia. Daptomycin was shown to interact in vitro with pulmonary surfactant, resulting in inhibition of antibacterial activity. This effect was specific to daptomycin and consistent with its known mechanism of action. This represents the first example of organ-specific inhibition of an antibiotic.

We studied the in vitro emergence of resistance to daptomycin using three methods: spontaneous resistance incidence, serial passage in the presence of increasing drug concentrations, and chemical mutagenesis. (12,19,25,26,28). The mechanism of action, while not yet fully elucidated, appears to involve disruption of bacterial plasma membrane function (1-5). Daptomycin was efficacious in phase 2 clinical trials in skin and soft tissue infection and bacteremia (28). Additional phase 2 and 3 clinical studies are ongoing.Results from in vitro studies and clinical trials indicate that resistance to daptomycin is rare (16,28; P. Courvalin, personal communication; G. W. Kaatz, personal communication). However, one group has reported relatively high resistance rates in vitro (18). In an effort to resolve this discrepancy, we determined spontaneous resistance rates and analyzed technical factors influencing those rates. In addition, the possible utility of resistant mutants in pinpointing daptomycin's mechanism of action prompted us to isolate and analyze resistant organisms.(Portions of this work were presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy [N. Oliver, T. Andrew, T. Li, and J. Silverman, poster F-117], San Diego, Calif., 24 to 27 September 1998.) MATERIALS AND METHODSStrains, media, and antibiotics. Bacteria were propagated at 37°C. Staphylococci were grown in Mueller-Hinton broth (MHB; Becton, Dickinson, Cockeysville, Md.), enterococci in brain heart infusion (Becton, Dickinson), and S. pneumoniae in Todd-Hewitt broth plus 5% horse serum. MIC testing was performed according to NCCLS guidelines for broth microdilution (23) except that all cultures were grown at 37°C. In addition, daptomycin MICs were determined using MHB supplemented with 50 mg of Ca 2ϩ per liter (MHBc). Modifications for determining nisin MICs were made as previously described (27). Vancomycin, ampicillin, gentamicin, and nisin were purchased from Sigma Chemical Company (St. Louis, Mo.). Complete defined media was obtained from JRH Biosciences (Lenexa, Kans.). Heterogeneity assay. Heterogeneous susceptibility was measured based on methods developed by de Lencastre et al. (8). Overnight cultures were plated at dilutions (made in MHBc) ranging from 10 0 to 10 Ϫ7 on Mueller-Hinton agar (MHA) or MHA plus 50 mg of CaCl 2 per liter containing twofold dilutions of daptomycin over the range 0.125 to 64 g/ml.Serial-passage mutagenesis. On day 1, MHBc containing daptomycin at 0.25, 0.5, 1, or 2 times the MIC was inoculated with S. aureus (strain Sa42) from a single colony. Cultures were incubated overnight at 37°C with shaking. From the highest concentration that supported growth, cultures were diluted 1:10,000 into fresh media plus daptomycin at twofold dilutions. This process was continued for 21 days or until three successive cultures failed to show any decrease in susceptibility. Putative mutants were colony purified for three generations on MHA, prior to further characterization.Chemical mutagenesis. N-Methyl-...

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The rising rates of antibiotic resistance accentuate the critical need for new antibiotics. Daptomycin is a new antibiotic with a unique mode of action and a rapid in vitro bactericidal effect against gram-positive organisms. This study examined the kinetics of daptomycin's bactericidal action against peritonitis caused by methicillinsusceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in healthy and neutropenic mice and compared this activity with those of other commonly used antibiotics. CD-1 mice were inoculated intraperitoneally with lethal doses of MSSA (Xen-29) or MRSA (Xen-1), laboratory strains transformed with a plasmid containing the lux operon, which confers bioluminescence. One hour later, the animals were given a single dose of daptomycin at 50 mg/kg of body weight subcutaneously (s.c.), nafcillin at 100 mg/kg s.c., vancomycin at 100 mg/kg s.c., linezolid at 100 mg/kg via gavage (orally), or saline (10 ml/kg s.c.). The mice were anesthetized hourly, and photon emissions from living bioluminescent bacteria were imaged and quantified. The luminescence in saline-treated control mice either increased (neutropenic mice) or remained relatively unchanged (healthy mice). In contrast, by 2 to 3 h postdosing, daptomycin effected a 90% reduction of luminescence of MSSA or MRSA in both healthy and neutropenic mice. The activity of daptomycin against both MSSA and MRSA strains was superior to those of nafcillin, vancomycin, and linezolid. Against MSSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than nafcillin or linezolid. Against MRSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than vancomycin or linezolid. The rapid decrease in the luminescent signal in the daptomycin-treated neutropenic mice underscores the potency of this antibiotic against S. aureus in the immune-suppressed host.

CB-183,315 is a novel lipopeptide antibiotic structurally related to daptomycin currently in phase 3 clinical development for Clostridium difficile-associated diarrhea (CDAD). We report here the in vitro mechanism of action, spontaneous resistance incidence, resistance by serial passage, time-kill kinetics, postantibiotic effect, and efficacy of CB-183,315 in a hamster model of lethal infection. In vitro data showed that CB-183,315 dissipated the membrane potential of Staphylococcus aureus without inducing changes in membrane permeability to small molecules. The rate of spontaneous resistance to CB-183,315 at 8؋ the MIC was below the limit of detection in C. difficile. Under selective pressure by serial passage with CB-183,315 against C. difficile, the susceptibility of the bacteria changed no more than 2-fold during 15 days of serial passages. At 16؋ the MIC, CB-183,315 produced a >3-log reduction of C. difficile in the time-kill assay. The postantibiotic effect of CB-183,315 at 8؋ the MIC was 0.9 h. At 80؋ the MIC the postantibiotic effect was more than 6 h. In the hamster model of CDAD, CB-183,315 and vancomycin both demonstrated potent efficacy in resolving initial disease onset, even at very low doses. After the conclusion of dosing, CB-183,315 and vancomycin showed a similar dose-and time-dependent pattern with respect to rates of CDAD recurrence.