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In Vitro Activities of CB-183,315, Vancomycin, and Metronidazole against 556 Strains of Clostridium difficile, 445 Other Intestinal Anaerobes, and 56 Enterobacteriaceae Species

ABSTRACT MICs of CB-183,315, a novel lipopeptide antibiotic, vancomycin, and metronidazole were determined for intestinal anaerobes and Enterobacteriaceae. The MIC90s for Gram-negative anaerobes were >8,192, 8,192, and 4 μg/ml for CB-183,315, vancomycin, and metronidazole, respectively. Against Enterobacteriaceae, the MIC90s were >8,192 μg/ml, 1,024 μg/ml, and 1,024 μg/ml, respectively. The CB-183,315 MIC90 for Clostridium difficile was 0.5 μg/ml. Its lack of activity against normal fecal organisms makes it a promising new agent for treating C. difficile.

C lostridium difficile infection (CDI) is the most common cause of health care-related diarrhea, usually associated with prior exposure to antimicrobial agents. The current theory is that the antibiotics disrupt the normal fecal microbiota, changing its complexity and diversity as a primary event, with the subsequent acquisition of toxigenic C. difficile as a secondary event in the development of the disease (6,10,13). CDI has increased in prevalence and severity during the last decade, resulting in increased mortality and complications, recurrent disease, and prolonged hospital stays (7,17). Non-health-care-related cases of CDI are now being reported (7,16). Treatment options have been limited by the unsatisfactory efficacy of current therapeutic agents, the high recurrence rates of disease, the disruption of normal intestinal microbiotas, and the colonization by vancomycin-resistant enterococci (VRE) (1,2,14). Although a recently introduced macrocyclic antibiotic, fidaxomicin, shows equivalent primary cure rates and reduced recurrence rates for CDI compared to those of vancomycin (15), it shows rates equivalent to those of vancomycin against restriction endonuclease analysis (REA) type BI strain-associated disease.
CB-183,315 is a novel lipopeptide antibiotic with Grampositive activity which is bactericidal against C. difficile. To study the spectrum of its activity, including its effect on the normal components of fecal microbiotas, we determined the MICs of CB-183,315 and vancomycin against C. difficile and other intestinal anaerobes, as well as against Escherichia coli, Klebsiella spp., and Enterobacter spp. For comparison, metronidazole was tested against the Gram-negative anaerobes and the Enterobacteriaceae spp.
The C. difficile strains were recent clinical isolates (2005 to 2008) recovered from stools of patients with CDI. The other isolates were obtained from stool or infection-containing organisms presumed to be of intestinal origin, identified by standard methods (12) and occasionally by 16S rRNA gene sequence analysis (20), and stored in 20% skim milk at Ϫ70°C.
MIC values were determined by the agar dilution method according to CLSI procedures (4). Vancomycin and metronidazole laboratory standard powders were obtained from Sigma (St. Louis, MO), and CB-183,315 was provided by Cubist Pharmaceuticals, Inc. (Lexington, MA). All assay media for testing CB-183,315 were supplemented with a final concentration of 50 mg/ liter calcium, where Ca 2ϩ concentrations were confirmed by Laboratory Specialists, Inc. (Westlake, OH).
The Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp. strains were tested using Mueller-Hinton agar and incubated at 37°C in ambient air, except for strains with metronidazole, which were incubated in the anaerobic chamber (4).
Quality control strains included Clostridium difficile ATCC 700057, Bacteroides fragilis ATCC 25285, Staphylococcus aureus ATCC 29213, and E. coli ATCC 25922 and were used each day of testing with the relevant set of test organisms.
Time-kill studies were carried out on one REA type BI strain (NAP1, ribotype 027) and one REA type Y strain (NAP4, ribotype 014) (3). CB-183,315 and vancomycin were prepared at 2, 4, and 8 times their MICs for each strain in supplemented Brucella broth. A drug-free growth control tube was included. Tubes were inoculated with ϳ10 6 CFU/ml, placed on a shaker, and assayed at 0, 2, 4, 8, and 24 h of incubation at 37°C. Table 1 shows the ranges, MIC 50 s, and MIC 90 s for the major groups of organisms. The CB-183,315 MIC range for C. difficile was 0.06 to 2.0 g/ml, with a MIC 90 of 0.5 g/ml; other Grampositive strains were inhibited by 0.03 to 16 g/ml of CB-183,315. The MIC 90 s of CB-183,315 for the Bacteroides fragilis group, Prevotella spp., Gram-negative cocci (Veillonella spp. and Acidaminococcus spp.), E. coli, Enterobacter spp., and Klebsiella spp. were all greater than 8,192 g/ml. The MIC 90 s were 8,192 g/ml and 2,048 g/ml for fusobacteria and Porphyromonas spp., respectively. In contrast, vancomycin had equally high MIC 90 s of Ͼ8,192 g/ml against only the fusobacteria and the Gram-negative cocci; the B. fragilis group was inhibited by vancomycin at a MIC 90 of 128 g/ml (range, 32 to 256 g/ml), Porphyromonas spp. were inhibited by vancomycin at a MIC 90 of 4 g/ml, and Prevotella spp. were inhibited at a MIC 90 of 256 g/ml of vancomycin. Vancomycin MICs ranged from 64 to 1,024 g/ml for E. coli and 256 to 1,024 g/ml for Enterobacter and Klebsiella species. Metronidazole was active against the Gram-negative anaerobes at 0.06 to 8 g/ml but showed poor activity against the Enterobacteriaceae, with MICs ranging from 32 to1,024 g/ml.
The time-kill studies showed that CB-183,315 at 4 and 8 times the MIC resulted in a 3-log 10 reduction in the colony count for both REA types tested after 24 h of incubation. Vancomycin produced similar results.
CB-183,315 reaches a concentration of 6,494 Ϯ 3,104 g/g of feces after a 1-g twice-a-day (b.i.d.) dose at day 5 of a 14-day course (Cubist Pharmaceuticals, unpublished data), a concentration which is lower than the high MICs (Ͼ8,192 g/ml) demonstrated for Bacteroides and other groups of organisms of the intes-tinal anaerobes; thus, CB-183,315 is likely to spare many of these important members of the normal microbiota. In contrast, vancomycin at a dose of 250 mg four times a day (q.i.d.) results in fecal levels that are generally above 2,000 g/ml (11). Vancomycin thus has the potential for killing or inhibiting much of the normal aerobic and anaerobic fecal microbiotas. Moreover, oral vancomycin was shown to decrease or suppress Bacteroides in volunteers while increasing the occurrence of vancomycin-resistant enterococci (8,13). In contrast, fidaxomicin, a narrow-spectrum macrocycle, displayed less impact on the Bacteroides populations in patients treated for CDI (13), an observation which has been postulated as one reason for the associated lower relapse rate.