1 Departments of Pathology (Clinical Microbiology), Hershey Medical Center, Hershey, PA 17033; 2 Case Western Reserve University, Cleveland, OH 44106, USA
Received 3 October 2001; returned 20 January 2002; revised 8 February 2002; accepted 2 April 2002
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Abstract |
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Introduction |
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Although development of an effective vaccine against Haemophilus influenzae type b has led to the disappearance of the organism in many parts of the world, untypeable H. influenzae strains have not been affected.5,6 These organisms together with Streptococcus pneumoniae and Moraxella catarrhalis are considered the leading causes of acute exacerbations of chronic bronchitis and other respiratory tract infections.5,6 Drugs used clinically in the empirical treatment of community-acquired respiratory tract infections currently include ß-lactams, macrolides (and azalides) and the new broad-spectrum quinolone group.
Cefditoren is an oral cephalosporin with excellent in vitro activity against penicillin-susceptible, -intermediate and -resistant pneumococci. Because of its excellent concomitant in vitro activity against H. influenzae and M. catarrhalis716 this compound shows promise for empirical treatment of otitis media and respiratory tract infections such as acute exacerbations of chronic bronchitis, and has been used successfully in Japan for this purpose for several years.
The current study attempted to shed further light on the antibacterial activity of cefditoren by comparing it with that of cefdinir, cefuroxime, cefprozil, cefpodoxime, cefixime, azithromycin, clarithromycin, ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin against a spectrum of pneumococci with differing drug susceptibilities and against 160 H. influenzae strains. In addition, the above oral cephalosporins were tested against 12 pneumococci and nine H. influenzae by timekill assay. A study was also undertaken to examine the ability of cefditoren, compared with co-amoxiclav, cefuroxime, cefprozil and azithromycin, to select resistant mutants in S. pneumoniae.
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Materials and methods |
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The pneumococci tested comprised 90 penicillin-susceptible (MICs 0.06 mg/L), 63 penicillin-intermediate (MICs 0.1251.0 mg/L) and 97 penicillin-resistant (MICs
2.0 mg/L) strains. Of these, 54 had raised quinolone MICs (ciprofloxacin MICs
4.0 mg/L) and 166 were macrolide resistant. A subset of 12 pneumococcal strains (four penicillin-susceptible, four intermediate and four resistant) were used in timekill studies. Of the 160 H. influenzae strains, 85 were ß-lactamase positive. Nine H. influenzae strains (three ß-lactamase positive) were selected for timekill testing. For resistance selection studies, 12 pneumococci (four penicillin-susceptible, four intermediate and four resistant) were tested.
Microdilution MIC
MICs were determined by the broth microdilution method recommended by the NCCLS,17 using cation-adjusted MuellerHinton broth (BBL Microbiology Systems) supplemented with 5% lysed horse blood for pneumococci and freshly made Haemophilus Test Medium (HTM) for H. influenzae. Frozen trays were prepared commercially by Trek Diagnostics (Westlake, OH, USA). Standard quality control strains3,17 were included in each run.
Timekill testing
For timekill studies, tubes containing 5 mL cation-adjusted MuellerHinton broth (Difco Laboratories) supplemented with 5% lysed horse blood (pneumococci) or freshly made HTM (H. influenzae) with doubling antibiotic concentrations were inoculated with 5 x 1055 x 106 cfu/mL and incubated at 35°C in a shaking water bath. Dilutions required to obtain the correct inoculum were determined by prior viability studies using each strain.18,19 Growth controls with inoculum but no antibiotics were included with each experiment.18,19
Viability counts of antibiotic-containing suspensions were tested by plating 10-fold dilutions of 0.1 mL aliquots from each tube in sterile MuellerHinton broth on to trypticase soy agar with 5% sheep blood (pneumococci) (BBL Microbiology Systems) or chocolate agar (H. influenzae) (BBL Microbiology Systems). Plates were incubated for up to 72 h. Counts were carried out on plates yielding 30300 colonies. The lower limit of colony count sensitivity was 300 cfu/mL.18,19
Timekill assays were analysed by determining the number of strains that yielded a log10 cfu/mL of 1, 2 and 3 at 3, 6, 12 and 24 h, compared with counts at time 0 h. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by
3 log10 cfu/mL (99.9%) at each of the time periods, and bacteriostatic if the inoculum was reduced by 0 to <3 log10 cfu/mL.18,19
Multistep mutation analysis
Glass tubes with 1 mL of cation-adjusted MuellerHinton broth (Difco Laboratories) supplemented with 5% lysed horse blood and containing two-fold increasing concentrations of antibiotic in the range 0.125 x to 16 x MIC were inoculated with c. 5 x 105 cfu/mL. Tubes were incubated at 35°C for 24 h. Daily passages were then performed for 50 days by subculturing 10 µL from the tube nearest the MIC (usually one to two dilutions below) that had the same turbidity as the antibiotic-free controls. Periodically for some mutants, an aliquot from a tube used as an inoculum was frozen in double-strength skimmed milk at 70°C for later analysis. When an MIC for a strain increased eight-fold, irrespective of the number of subcultures, passaging was stopped and strains were subcultured in antibiotic-free medium for 10 serial passages. A maximum of 50 serial passages in antibiotic were performed. The susceptibilities of resistant mutants to all compounds were tested by MIC determination.2022
Single-step mutation analysis
Frequency of spontaneous single-step mutation was determined by spreading c. 1 x 1010 cfu/mL in 100 µL aliquots on 5% sheep bloodbrainheart infusion agar plates containing 1 x, 2 x, 4 x and 8 x MIC of each compound. Plates were incubated in 5% CO2 (to encourage growth) at 35°C for 4872 h. The resistance frequency was calculated as the number of resistant colonies per inoculum.21
Identity of resistant clones
To determine whether resistant clones were identical to parents, strains were characterized by serotyping and PFGE as described previously.2022
Mechanism of resistance in resistance selection studies. Resistant clones were tested for macrolide resistance mechanisms as described previously.20,22,23 For ß-lactam resistance in mutant clones of S. pneumoniae, penicillin-binding proteins (PBPs) 1a, 2x and 2b of S. pneumoniae were analysed by the following procedure: template DNA for PCR was prepared using the Prep-A-Gene kit (Bio-Rad, Hercules, CA, USA) as recommended by the manufacturer. A 1.2 kb segment of each of pbp1a, pbp2b and pbp2x was amplified by PCR using primers and cycling conditions as described previously.2426
The results of DNA sequencing were aligned using Vector NTI 6.0 (Infomax, Inc., Bethesda, MD, USA) to compare the DNA and amino acid sequences from the parent and mutant strains.
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Results |
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For the 12 strains tested for multistep mutations (Table 3), MICs (mg/L) were as follows: cefditoren, 0.0160.06 (penicillin susceptible), 0.030.125 (penicillin intermediate), 1.0 (penicillin resistant); co-amoxiclav, 0.0160.03 (penicillin susceptible), 0.060.125 (penicillin intermediate), 2.0 (penicillin resistant); cefuroxime, 0.030.06 (penicillin susceptible), 0.250.5 (penicillin intermediate), 8.0 (penicillin resistant); cefprozil, 0.1250.25 (penicillin susceptible), 0.51.0 (penicillin intermediate), 16.0 (penicillin resistant); azithromycin, 0.0160.03 (penicillin susceptible), 0.064.0 (penicillin intermediate), 1.04.0 (penicillin resistant). After 50 subcultures in sub-MIC concentrations of cefditoren, MICs rose by one dilution for two strains, by two dilutions for four strains and by three dilutions for one strain. In comparison, selection with co-amoxiclav led to a one-dilution increase in MIC for six strains and a two-dilution increase for one strain, whereas selection with cefuroxime led to a one-dilution increase for one strain, a two-dilution increase for six, a three-dilution increase for one and an increase of four or more dilutions for two strains. For the remaining strains, no increase in ß-lactam MIC was found even after 50 subcultures. In comparison, cefprozil led to a one-dilution increase for two strains, a two-dilution increase for eight and a three-dilution increase for two strains. Azithromycin led to an increase of two dilutions for one strain, three dilutions for two and four or more dilutions for nine strains. Results of resistance mechanisms can be seen in Table 3. Most resistant clones appeared with azithromycin. Abnormalities in ß-lactam-resistant clones occurred in pbp1a and/or pbp2x in two strains (Table 3).
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Discussion |
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MIC results of other compounds tested, including amoxicillin, co-amoxiclav and other oral cephalosporins, were similar to those described previously.3,27,28 Amoxicillin had lower MICs for pneumococci than currently available oral cephalosporins whereas cefixime and cefpodoxime were the most active oral ß-lactams against H. influenzae.
Multistep resistance selection studies in pneumococci revealed that cefditoren, co-amoxiclav and cefprozil failed to select for resistant mutants in 12 penicillin-susceptible, -intermediate and -resistant strains of S. pneumoniae after 50 subcultures. In comparison, cefuroxime selected for mutants with raised MICs in 2/12 strains and azithromycin in 9/12 strains. The reason for the low rate of selection of resistant mutants in S.pneumoniae by ß-lactams (especially co-amoxiclav and cefditoren) compared with macrolides and quinolones 2023 is unknown at present but may be related to (i) differing mechanisms of action between the three antibiotic groups; (ii) differing affinities of co-amoxiclav and cefditoren for penicillin-binding proteins compared with cefuroxime and cefaclor, which yield more clones with raised ß-lactam MICs by this method.20 These aspects are currently under investigation.
Results of single-step mutations in the same 12 pneumococcal strains showed that cefditoren, at the MIC, selected for spontaneous mutants at a lower rate than other drugs tested in five of 12 organisms. In comparison, the other drugs and number of times they had the lowest mutation rate at the MIC were: co-amoxiclav and cefuroxime, 4; cefprozil, 1; azithromycin, 0.
In summary, cefditoren exhibited the lowest MICs and best timekill kinetics against a variety of drug-susceptible and -resistant S. pneumoniae as well as H. influenzae strains, compared with other oral cephalosporins, and compared very favourably with co-amoxiclav. In addition, MICs for H. influenzae (ß-lactamase-positive and -negative) were low compared with cefpodoxime and cefixime. Cefditoren as well as co-amoxiclav and cefprozil did not select resistant mutants of S. pneumoniae even after 50 subcultures, and cefditoren yielded the lowest frequency of spontaneous mutants in five of 12 pneumococcal strains tested. Results indicate that cefditoren is a promising drug for the treatment of community-acquired respiratory tract infections, and that, like co-amoxiclav (and unlike other oral cephalosporins), it may not have the potential to select resistant pneumococci.
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Acknowledgements |
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Footnotes |
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References |
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