Activities of Ceftobiprole, a Novel Broad-Spectrum Cephalosporin, against Haemophilus influenzae and Moraxella catarrhalis

MIC determinations.A total of 321 strains of H. influenzae were surveyed. These were isolated between 1999 and 2002, predominantly from sputum, bronchial aspirate, blood, and cerebrospinal fluid (the last two sources from countries which do not use the H. influenzae type b vaccine), and were comprised of 262 β-lactamase-positive strains (including two β-lactamase-positive amoxicillin-clavulanate-resistant [BLPACR] strains), 40 β-lactamase-negative strains, and 19 BLNAR strains. The H. influenzae panel also included nine quinolone-nonsusceptible strains, two macrolide-hypersusceptible strains, and one macrolide-hyperresistant strain (31, 32). The 49 M. catarrhalis strains were comprised of 40 β-lactamase producers and nine β-lactamase nonproducers. Strains were stored at −70°C in double-strength skim milk (Difco Laboratories, Detroit, Mich.) before MIC testing and periodically examined for purity by cultivation and Gram staining.

β-Lactamase testing was performed using nitrocefin disks (Cefinase; BBL Microbiology Systems, Inc., Cockeysville, Md.).

Susceptibility testing was performed by the microdilution broth method according to CLSI guidelines (28) using freshly prepared Haemophilus test medium (HTM) in 96-well microtiter plates manufactured by TREK, Inc. (Westlake, Ohio). Ceftobiprole powder was a gift from Basilea Pharmaceutica AG (Basel, Switzerland), whereas other compounds were obtained from their respective manufacturers. Inocula, prepared from 18-h chocolate agar plates (BBL, Cockeysville, Md.) by direct colony suspension, contained 3 × 10⁴ to 7 × 10⁴ CFU/well. Quality control strains H. influenzae ATCC 10211, H. influenzae ATCC 49247, and H. influenzae ATCC 49766 were included in each set of experiments. Microtiter plates were incubated in ambient air at 35°C.

Time-kill studies.For time-kill profiling 10 H. influenzae strains with the following phenotypes were selected: four β-lactamase positive, two β-lactamase negative, two BLPACR, and two BLNAR. Of these, six strains with amoxicillin MICs of >32 μg/ml were not tested with amoxicillin. Two β-lactamase-positive M. catarrhalis strains also were examined by time-kill assays.

Glass tubes containing 5 ml of freshly prepared HTM containing doubling antibiotic concentrations were inoculated with 5 × 10⁵ to 5 × 10⁶ CFU/ml and incubated at 35°C in a shaking water bath. Viability counts of antibiotic-containing suspensions and controls lacking antibiotic were obtained at 0, 3, 6, 12, and 24 h by plating 10-fold dilutions (HTM) of 0.1-ml aliquots from each tube onto chocolate agar plates, which were incubated for up to 48 h in 5% CO₂ at 35°C. Colony counts were performed on plates yielding 30 to 300 colonies; the lower limit of sensitivity of colony counts was 300 CFU/ml. The number of strains yielding a Δ(log₁₀ CFU/ml) of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 3, 6, 12, and 24 h relative to the inoculum size was determined. A given concentration of antibiotic (expressed as a multiplicity of MIC) was considered bactericidal if it reduced the inoculum viable count by ≥3 log₁₀ CFU/ml or bacteriostatic if it reduced the inoculum viable count by <3 log₁₀ CFU/ml during a specified time period.

Multistep selection studies.For resistance selection studies eight H. influenzae isolates (six β-lactamase producers including a BLPACR strain and two β-lactamase nonproducers including a BLNAR strain) and two M. catarrhalis isolates (both β-lactamase positive) were chosen. Serial passage of each strain (initial inoculum size, ∼10⁸ CFU/ml) was performed daily using freshly prepared HTM supplemented with increasing subinhibitory concentrations of a given antibiotic. For each subsequent daily passage the inoculum was taken from the tube nearest the MIC, usually 1 or 2 log₂ dilution steps below the MIC, which had approximately the same turbidity as antibiotic-free controls. Strains were passaged until MICs of >64 μg/ml were obtained, up to a maximum of 50 serial passages, at which time subculturing in the presence of antibiotic was discontinued, and selected resistant/nonsusceptible clones underwent 10 daily passages on chocolate agar without antibiotics. The identity of parental strains and their derived clones was confirmed by pulsed-field gel electrophoresis using a CHEF DR III apparatus (Bio-Rad, Hercules, CA). MICs for each resistant clone for each drug were confirmed by microdilution broth assay. Resistance mechanisms for some parental strains and derived clones were determined as described below.

Mechanisms of resistance.The presence of mutations in ribosomal L4 and L22 proteins and in 23S rRNA (for macrolide-resistant clones) and in quinolone resistance-determining regions (for moxifloxacin-resistant clones) was examined using primers and conditions as described previously (6, 31). After PCR amplification products were purified using a QIAquick PCR purification kit (QIAGEN, Valencia, Calif.). Nucleotide sequences were obtained using a CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA).

Single-step selection studies.Selection frequencies of clones resistant to 2×, 4×, and 8× the MIC of antibiotics were determined by spreading 10⁹ to 10¹¹ CFU (in 100 μl) onto plates of either HTM (H. influenzae) or Mueller-Hinton agar supplemented with 5% sheep blood (M. catarrhalis). After incubation at 35°C in 5% CO₂ for 48 h, resistant colonies were confirmed by replica plating onto medium containing antibiotics. Resistance frequencies were calculated as the number of resistant colonies per inoculum (21).

Time-kill studies.Macrodilution broth MICs for the 12 strains chosen for time-kill studies are listed in Table 2, and summaries of time-kill results for the H. influenzae strains surveyed are presented in Table 3. Ceftobiprole was bactericidal against 7 of 10 H. influenzae strains at 1× MIC and against all 10 strains at 2× MIC by 24 h. Amoxicillin-clavulanate, ceftriaxone, cefpodoxime, azithromycin, telithromycin, and moxifloxacin produced similar time-kill profiles relative to their MICs. Bactericidal activity at 2× MIC after 24 h was observed for tetracycline against 6 of 10 strains and for amoxicillin against two of four strains tested.

Time-kill analyses of two β-lactamase-positive M. catarrhalis strains (data not shown) found that ceftobiprole and amoxicillin were bactericidal against one strain at 4× MIC by 24 h. Amoxicillin-clavulanate, ceftriaxone, cefpodoxime, azithromycin, telithromycin, and moxifloxacin were all bactericidal against both M. catarrhalis strains at 2× MIC after 24 h. Tetracycline was bactericidal against one strain at 2× MIC and against both M. catarrhalis strains at 4× MIC after 24 h.

Multipassage resistance selection studies.The initial MIC ranges for parental strains were as follows: ceftobiprole, 0.03 to 1 μg/ml; amoxicillin-clavulanate, 0.5 to 8 μg/ml; ceftriaxone, 0.004 to 0.25 μg/ml; azithromycin, 0.25 to 2 μg/ml; telithromycin, 0.5 to 2 μg/ml; and moxifloxacin, 0.016 to 0.06 μg/ml (Table 4).

After 50 serial passages in the presence of ceftobiprole MICs for the eight H. influenzae strains surveyed remained constant for five strains and increased 1 to 2 log₂ dilution steps for the other three strains; the highest MIC recorded was 1 μg/ml. Ceftriaxone selection likewise led to MIC increases of 0 to 2 log₂ dilution steps, the highest ceftriaxone MIC recorded being 0.25 μg/ml. No increase in MICs was observed for strains subcultured in amoxicillin-clavulanate. During serial passage seven of eight H. influenzae strains gave rise to azithromycin-nonsusceptible clones according to CLSI breakpoint criteria (7); two of these clones, recovered after 34 to 37 passages, had undergone MIC increases of 1 to >64 μg/ml. In contrast, only three of eight strains evolved resistance to telithromycin (2 to 16 μg/ml, 1 to 32 μg/ml, and 0.5 to >64 μg/ml) according to CLSI breakpoint criteria. Selection with moxifloxacin produced only one clone nonsusceptible to this fluoroquinolone (0.06 to 32 μg/ml), though MIC increases as high as 32-fold were found for the remaining seven H. influenzae clones passaged in the presence of moxifloxacin. Mutations in 23S rRNA and/or in ribosomal proteins L4 and L22 were identified in three of six azithromycin-nonsusceptible and telithromycin-resistant clones (Table 4). Sequencing of five moxifloxacin-nonsusceptible clones (MIC, 0.5 to 32 μg/ml) revealed mutations in GyrA, GyrB, and/or ParC in three clones (Table 4).

The MIC for azithromycin of one azithromycin-nonsusceptible clone, derived from H. influenzae parental strain 153-008, was somewhat unstable, dropping from >64 μg/ml to 32 μg/ml after 10 passages in drug-free medium (Table 5), though MICs for other antibiotics in the drug panel either remained unchanged or rose by 1 to 2 log₂ dilution steps. After 10 passages on drug-free medium, the azithromycin-nonsusceptible clone derived from H. influenzae parental strain 110-061 showed no MIC changes for azithromycin, ceftobiprole, ceftriaxone, or amoxicillin-clavulanate outside a single log₂ dilution step, though its moxifloxacin MIC dropped from 1 μg/ml to 0.125 μg/ml (Table 5). During passage on drug-free medium the MIC for telithromycin of this clone rose from 4 μg/ml to >32 μg/ml. We have no explanation for this phenomenon, which is currently under investigation.

After 50 serial passages in the presence of antibiotic, MICs for the two M. catarrhalis strains either remained stable or increased by 1 log₂ dilution step (Table 4). Selection by moxifloxacin and azithromycin did not lead to increases in MICs compared to the parental strains (0.06 μg/ml for moxifloxacin and 0.03 μg/ml for azithromycin).

Single-passage selection studies.Single-step selection did not identify any H. influenzae and M. catarrhalis clones with enhanced resistance to ceftobiprole; selection frequencies for all strains ranged between <5.0 × 10⁻¹⁰ and <1.3 × 10⁻¹¹ at 2× MIC and at 8× MIC (Table 6). In contrast, amoxicillin-clavulanate, ceftriaxone, and moxifloxacin each selected for clones with MICs four- to eightfold higher than those of the parental strains. Selection frequencies for these drugs were as follows: amoxicillin-clavulanate, >3.6 × 10⁻⁶ to <1.0 × 10⁻¹⁰ (2× MIC) and >3.6 × 10⁻⁷ to < 1.0 × 10⁻¹⁰ (8× MIC); ceftriaxone, 7.5 × 10⁻⁷ to <8.3 × 10⁻¹¹ (2× MIC) and <8.3 × 10⁻¹⁰ to <8.3 × 10⁻¹¹ (8× MIC); moxifloxacin, 3.3 × 10⁻⁸ to <3.2 × 10⁻¹⁰ (2× MIC) and 3.3 × 10⁻⁹ to <2.6 × 10⁻¹⁰ (8× MIC). The greatest increases in MICs (4- to >32-fold) were observed for azithromycin and telithromycin, with selection frequencies of 1.3 × 10⁻⁸ to <2.9 × 10⁻¹⁰ (2× MIC) and 2.0 × 10⁻¹⁰ to <1.3 × 10⁻¹⁰ (8× MIC) for azithromycin and 1.3 × 10⁻⁸ to <1.0 × 10⁻¹⁰ (2× MIC) and 2.3 × 10⁻¹⁰ to <1.0 × 10⁻¹⁰ (8× MIC) for telithromycin (Table 6).