Activity of cefditoren against respiratory pathogens

Catherine L. Clark1, Kensuke Nagai1, Bonifacio E. Dewasse1, Glenn A. Pankuch1, Lois M. Ednie1, Michael R. Jacobs2 and Peter C. Appelbaum1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The activity of cefditoren and six other cephalosporins was tested against 250 pneumococci, including strains resistant to macrolides and quinolones. Cefditoren gave the lowest MICs, with MIC50 and MIC90 values of <=0.016/0.03, 0.125/0.5 and 0.5/2.0 mg/L for penicillin-susceptible, -intermediate and -resistant pneumococci, respectively. A time–kill study of 12 pneumococcal strains with varying drug susceptibilities showed that cefditoren, at 2 x MIC, gave 99% killing of all strains after 12 h, with 99.9% killing after 24 h. Other cephalosporins gave similar kill kinetics but at higher concentrations. Against 160 Haemophilus influenzae, cefditoren had the lowest MICs (MIC50 and MIC90 both <=0.016 mg/L), irrespective of ß-lactamase production. Time–kill studies of cefditoren compared with five other oral cephalosporins showed that cefditoren, at 8 x MIC, was bactericidal against 8/9 strains and gave 90% killing of all strains at the MIC after 12 h. Activity was bactericidal (99.9% killing) after 24 h with all drugs tested. Multistep studies of four penicillin-susceptible, four penicillin-intermediate and four penicillin-resistant strains showed that cefditoren, co-amoxiclav and cefprozil did not select for resistant mutants after 50 subcultures, compared with cefuroxime and azithromycin, where resistant mutants were selected in two and nine strains, respectively. Single-step mutation studies showed that cefditoren, at the MIC, had the lowest frequency of spontaneous mutants compared with other drugs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Community-acquired respiratory tract infections caused by pneumococci with decreased susceptibility to penicillin and other ß-lactam and non-ß-lactam antibiotics have become a problem throughout the USA and elsewhere.13 A recent survey reported that 50.4% of 1476 pneumococcal strains had raised penicillin MICs, and an overall macrolide resistance rate of 33%.3 The problem of drug-resistant pneumococci is complicated by the ability of this organism to spread from country to country, and from continent to continent.4

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 time–kill 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

The pneumococci tested comprised 90 penicillin-susceptible (MICs <= 0.06 mg/L), 63 penicillin-intermediate (MICs 0.125–1.0 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 time–kill studies. Of the 160 H. influenzae strains, 85 were ß-lactamase positive. Nine H. influenzae strains (three ß-lactamase positive) were selected for time–kill 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 Mueller–Hinton 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.

Time–kill testing

For time–kill studies, tubes containing 5 mL cation-adjusted Mueller–Hinton 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 105–5 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 Mueller–Hinton 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 30–300 colonies. The lower limit of colony count sensitivity was 300 cfu/mL.18,19

Time–kill assays were analysed by determining the number of strains that yielded a {Delta}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 Mueller–Hinton 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 blood–brain–heart 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 48–72 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In MIC studies against 250 pneumococci, cefditoren gave the lowest ß-lactam MICs, with MIC50 and MIC90 values of <=0.016/0.03, 0.125/0.5 and 0.5/2.0 mg/L for penicillin-susceptible, -intermediate and -resistant strains, respectively. Cefuroxime, the next most active cephalosporin, gave corresponding values of <=0.03/0.125, 0.5/4.0 and 4.0/8.0 mg/L (Table 1). Time–kill studies showed that cefditoren, at 2 x MIC (<=1.0 mg/L), was bactericidal against all 12 strains after 24 h with 99% killing of all strains at 2 x MIC after 12 h. Other cephalosporins gave similar kinetics, but at higher concentrations.


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Table 1. Microdilution MICs (mg/L) for 250 pneumococcal strains
 
MICs for the 160 H. influenzae strains tested are presented in Table 2. Cefditoren had MIC50 and MIC90 values of <=0.016 mg/L, irrespective of whether the strain tested produced ß-lactamase. Four ß-lactamase-negative strains of H. influenzae for which ß-lactam MICs were raised (cefditoren 0.06–0.5 mg/L, cefixime and cefpodoxime 0.125–1.0 mg/L, cefdinir 2.0–4.0 mg/L, cefuroxime 2.0–8.0 mg/L, cefprozil 2.0–16.0 mg/L), probably belonged to the ß-lactamase-negative ampicillin-resistant (BLNAR) phenotype.


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Table 2.  MIC50/90 (mg/L) for 160 H. influenzae strains tested
 
In time–kill studies of cefditoren and five other oral cephalosporins against nine H. influenzae strains showed that cefditoren, at 8 x MIC, was bactericidal against eight of the nine strains tested and gave 90% killing of all strains at the MIC after 12 h. Significant bactericidal activity (99.9% killing) occurred after 24 h with all drugs tested. No difference was observed, relative to the MIC, in kill kinetics against BLNAR and ß-lactamase-positive H. influenzae strains compared with ß-lactam-susceptible strains.

For the 12 strains tested for multistep mutations (Table 3), MICs (mg/L) were as follows: cefditoren, 0.016–0.06 (penicillin susceptible), 0.03–0.125 (penicillin intermediate), 1.0 (penicillin resistant); co-amoxiclav, 0.016–0.03 (penicillin susceptible), 0.06–0.125 (penicillin intermediate), 2.0 (penicillin resistant); cefuroxime, 0.03–0.06 (penicillin susceptible), 0.25–0.5 (penicillin intermediate), 8.0 (penicillin resistant); cefprozil, 0.125–0.25 (penicillin susceptible), 0.5–1.0 (penicillin intermediate), 16.0 (penicillin resistant); azithromycin, 0.016–0.03 (penicillin susceptible), 0.06–4.0 (penicillin intermediate), 1.0–4.0 (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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Table 3.  Multistep resistance selection results
 
Results of single-step resistance selection studies are presented in Table 4. The frequency of resistance selection at the MIC was the lowest for cefditoren in five of the 12 strains (<5.0 x 10–10 to 3.3 x 10–8). At 2 x, 4 x and 8 x MIC, cefditoren resulted in the lowest resistance frequency (<3.3 x 10–10 to <1.7 x 10–9, <3.3 x 10–10 to <1.3 x 10–9, <3.3 x 10–10 to <1.3 x 10–9) in three, two and two of 12 strains, respectively. In comparison, co-amoxiclav at the MIC yielded the lowest resistance frequency in four of the 12 strains (<5.0 x 10–10 to 4.2 x 10–6). At 2 x, 4 x and 8 x MIC co-amoxiclav resulted in the lowest resistance frequency (<3.3 x 10–10 to <5.0 x 10–10, <3.3 x 10–10 to <5.0 x 10–10, <3.3 x 10–10 to <5.0 x 10–10) in five, five and five strains, respectively. Equivalent values for cefuroxime at the MIC yielded the lowest resistance frequency in four of the 12 strains (<5.0 x 10–10 to <5.0 x 10–9). At 2 x, 4 x and 8 x MIC, cefuroxime resulted in the lowest resistance selection (all <5.0 x 10–10) in one, one and one strain, respectively. Cefprozil at the MIC yielded the lowest resistance frequency in one of the 12 strains (8.3 x 10–7). At 2 x, 4 x and 8 x MIC cefprozil resulted in the lowest resist-ance frequency (3.3 x 10–10 to <8.3 x 10–10, <3.3 x 10–10 to <1.0 x 10–9, <3.3 x 10–10 to <1.0 x 10–9) in two, five and five strains, respectively. Azithromycin at the MIC yielded the lowest resistance frequency in none of 12 strains. At 2 x, 4 x and 8 x MIC, azithromycin resulted in the lowest resist- ance frequency (<3.3 x 10–10 to <5.0 x 10–10, <3.3 x 10–10 to <5.0 x 10–10, <1.0 x 10–10 to <5.0 x 10–10) in two, six and eight strains, respectively.


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Table 4.  Frequency of single-step mutation for 12 S. pneumoniae
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study confirm the excellent in vitro activity of cefditoren compared with other oral ß-lactams against a broad range of pneumococci, irrespective of their resistance to ß-lactams, macrolides and quinolones.714 Results of MIC and time–kill testing show that cefditoren was also very active against a broader phenotypic spectrum of H. influenzae strains (including BLNAR strains) than had been tested previously,10,11 with low MICs and good kill kinetics. Although bactericidal activity was observed only after 24 h, results were similar to those of cefixime and cefpodoxime, two oral cephalosporins with recognized excellent clinical activity against H. influenzae.

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 time–kill 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.


    Acknowledgements
 
This study was supported by a grant from TAP Pharmaceutical Products, Inc., Lake Forest, IL, USA.


    Footnotes
 
* Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psu.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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