Selection of resistance of telithromycin against Haemophilus influenzae, Moraxella catarrhalis and streptococci in comparison with macrolides

Lorenzo Drago*, Elena De Vecchi, Lucia Nicola, Alberto Colombo and Maria Rita Gismondo

Laboratory of Clinical Microbiology, Department of Clinical Sciences ‘L. Sacco’ Teaching Hospital, University of Milan, Via GB Grassi 74, 20157 Milan, Italy

Received 23 February 2004; returned 16 April 2004; revised 29 April 2004; accepted 26 May 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: The in vitro abilities of telithromycin, azithromycin and clarithromycin to select for resistance were compared by testing isolates of Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and ß-haemolytic streptococci.

Methods: Five strains each of ß-lactamase-positive and ß-lactamase-negative H. influenzae, ß-lactamase-positive and ß-lactamase-negative M. catarrhalis, S. pneumoniae, ß-haemolytic group A, group C and group G streptococci and three strains of ß-lactamase-negative ampicillin-resistant H. influenzae were evaluated. Development of resistance was determined by multi-step and single-step methodologies. For multi-step studies, MIC values were determined after five serial passages on antibiotic-gradient plates and after 10 serial passages on antibiotic-free plates. Acquisition of resistance was defined as an increase of ≥4-fold from the starting MIC. In single-step studies, the rate of spontaneous mutations was calculated after a passage on plates containing antibiotics at concentrations equal to the highest NCCLS breakpoints.

Results: Azithromycin, clarithromycin and telithromycin gave a ≥4-fold increase in 20, 20 and 10 streptococcus strains, in 4, 5 and 0 H. influenzae strains and in 2, 7 and 4 M. catarrhalis strains, respectively. After 10 passages on antibiotic-free plates, 21/26 strains for azithromycin, 22/32 for clarithromycin and 1/14 for telithromycin maintained high MIC values. In single-step studies, the frequency of mutations was <10–10 for H. influenzae and M. catarrhalis for telithromycin, azithromycin and clarithromycin. Telithromycin induced mutations at a lower rate than azithromycin and clarithromycin in streptococcal strains.

Conclusion: Telithromycin showed a very limited ability to select for resistance in respiratory pathogens compared with azithromycin and clarithromycin.

Keywords: azithromycin , clarithromycin , ketolides , in vitro selection of resistance , respiratory pathogens


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibacterial selection for treatment of respiratory infections is generally empirical. Given the rapid spread of antibacterial resistance among respiratory pathogens, the choice of antibacterial agents is often complicated by the high resistance rates to macrolides and ß-lactams in respiratory pathogens.1 Moreover, the ability to rapidly induce resistance during treatment should also be considered, since it may be responsible for increase in resistance and treatment failure.

Ketolides belong to the macrolide-lincosamide-streptogramin B (MLSB) group, developed for the treatment of upper and lower respiratory tract infections, caused by common and atypical pathogens, including resistant streptococci.2 Compared with macrolides, the molecular structure of telithromycin is characterized by the absence of the cladinose group, which is probably linked to induction of resistance in macrolides.3

The aim of this study was to compare the ability of telithromycin to select for resistance in common respiratory pathogens compared with azithromycin and clarithromycin.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microorganisms

Five strains of S. pneumoniae, ß-haemolytic group A, C and G streptococci, ß-lactamase-positive (BLP) and ß-lactamase-negative (BLN) H. influenzae, BLP and BLN M. catarrhalis and three strains of ß-lactamase-negative ampicillin-resistant (BLNAR) H. influenzae, isolated from different patients at the Clinical Microbiology Laboratory of L. Sacco Teaching Hospital in Milan, were included in the study. The strains were stored at –80°C in brain heart infusion broth or Haemophilus test medium (HTM) broth, both supplemented with 10% glycerol before testing and checked for purity throughout the study by culture and Gram staining.

Drugs

Stock solutions of azithromycin (Pfizer, Rome, Italy), clarithromycin (Abbott Italy, Rome, Italy) and telithromycin (Aventis Pharma, Lainate, Italy) were prepared in 95% ethanol (azithromycin) and methanol (clarithromycin and telithromycin) at concentrations of 5120 mg/L and stored in aliquots at –20°C until use.

Determination of MIC

Antibiotic susceptibilities were determined using a broth microdilution method according to the NCCLS approved standard,4 incubating trays for 16–20 h in a CO2-enriched atmosphere. Mueller–Hinton broth supplemented with 5% lysed horse blood was used to assay streptococci, H. influenzae was tested with HTM broth, and cation-adjusted Mueller–Hinton broth was used for M. catarrhalis. Susceptibilities to azithromycin and clarithromycin were evaluated based on NCCLS breakpoints for S. pneumoniae, ß-haemolytic streptococci and H. influenzae.4 Activity of telithromycin against S. pneumoniae and H. influenzae was defined by NCCLS breakpoints, while breakpoints of S. pneumoniae were adopted for ß-haemolytic streptococci.5

Selection of resistant bacteria (multi-step)

The ability to select resistant bacteria was evaluated by serial passages on agar plates containing a linear gradient of the antibiotics under evaluation, as previously described.6 Gradients were prepared in Petri dishes, which were poured with two layers of agar. The bottom layer consisted of antibiotic-free agar, allowed to harden with the plate slanted sufficiently to cover the entire bottom. The top layer, added to the dish in the normal position, generally contained antibiotic at concentrations of about 4–8x MIC.

The media used were Columbia agar supplemented with 5% sheep blood for streptococci and M. catarrhalis and HTM agar for H. influenzae. An inoculum of 0.1 mL containing 1010 cfu was homogenously spread on each plate, and incubated for 48 h at 37°C in a CO2 enriched atmosphere. Colonies growing at the highest antibiotic concentration were sampled, checked for purity, grown overnight on antibiotic-free agar, and replated on new antibiotic-gradient plates. Bacteria were exposed to a maximum of five consecutive passages on the antibiotic gradients followed by 10 passages on antibiotic-free medium. MIC values were determined after the first, second and fifth passage in antibiotic-containing agar and after the tenth passage in antibiotic-free medium by the broth microdilution technique as described above.

Acquisition of resistance was defined as a ≥4-fold increase in MIC.

Mutational frequency (single-step)

The frequency of spontaneous single-step mutations was determined by spreading 0.1 mL from a bacterial suspension of about 1010 cfu/mL on antibiotic-free agar plates (after 10–7 and 10–8 dilutions to obtain colony counts ranging from 30 to 300 colonies) and on antibiotic-containing agar plates (undiluted inoculum). Columbia agar with 5% sheep blood was used for streptococci and M. catarrhalis and HTM agar was used for H. influenzae. NCCLS resistance breakpoints, when available, were used to define concentrations of azithromycin, clarithromycin and telithromycin. For ß-haemolytic streptococci, a concentration equal to that used for S. pneumoniae was chosen for telithromycin.5 Since NCCLS breakpoints for M. catarrhalis are not available, we arbitrarily chose to use concentrations used for H. influenzae.

Colonies grown after 48 h of incubation at 37°C in a CO2 atmosphere were counted. The frequency of mutation was calculated as the number of resistant colonies per inoculum.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selection of resistant bacteria (multi-step)

Results of gradient-plates resistance selection are summarized in Table 1.


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Table 1. Selection of resistance on antibiotic-gradient plates

 
Azithromycin seemed to be more rapid than the comparators at inducing a four-fold increase in MICs for streptococci, since after only one passage in gradient plates, 4/5 strains of S. pneumoniae, and 12/15 strains of ß-haemolytic streptococci showed such increments. After five passages, MICs of telithromycin increased ≥4-fold in two strains of S. pneumoniae and in eight strains of ß-haemolytic streptococci, whereas those of azithromycin and clarithromycin increased in all the streptococcal strains. After 10 passages in antibiotic-free medium, initial MIC values were recovered by all but one of the strains with increased MICs of telithromycin. In contrast, MICs obtained after the fifth passage with azithromycin and clarithromycin remained increased after 10 passages on antibiotic-free medium in most of the streptococci, being also above the resistant breakpoint of azithromycin and clarithromycin in 18 and 9 strains, respectively.

BLP H. influenzae were no more likely to select for resistance to macrolides than BLN strains. After five passages on antibiotic-containing plates, no ≥4-fold MIC increase occurred for telithromycin, while four and five strains showed MICs of azithromycin and clarithromycin increased by at least four-fold. After 10 passages in antibiotic-free medium, MIC values remained high in three strains for each antibiotic. At this stage, resistance to azithromycin and clarithromycin was detected in two strains.

After five passages, clarithromycin exposure caused a four-fold increase in MICs in more instances than azithromycin and telithromycin in BLN M. catarrhalis strains, while similar results were observed for clarithromycin and telithromycin for BLP M. catarrhalis. After passages in antibiotic-free medium, all strains recovered initial MIC values for azithromycin and telithromycin, while four strains maintained the increased MIC values for clarithromycin.

Mutational frequency (single-step)

Single-step mutation results are shown in Table 2. Clarithromycin and azithromycin produced the highest mutation rates, followed by telithromycin for S. pneumoniae and ß-haemolytic streptococci. Mutational rates were lower than 10–10 for all the tested strains of H. influenzae and M. catarrhalis.


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Table 2. Frequency of spontaneous mutations induced by azithromycin, clarithromycin and telithromycin

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present work, we assessed the ability of telithromycin to induce resistance compared with two macrolides. Our results for S. pneumoniae are consistent with data from other authors, confirming the limited reduction in susceptibility induced by telithromycin compared with macrolides.3,7 The increase in telithromycin MICs seems to be less stable than that of macrolides, being lost after serial subcultures in antibiotic-free medium and might represent a favourable aspect of telithromycin, if confirmed in in vivo studies.

Although ß-haemolytic streptococci remain widely susceptible to penicillin, resistance to macrolides is rather common. In this study, susceptibilities of these bacteria after exposure to antibiotics decreased more markedly for azithromycin and clarithromycin than for telithromycin both after one-step and multi-step selection. Moreover, compared with macrolides, the decrease in susceptibility induced by telithromycin was less pronounced, rather unstable, and lost after subculture in antibiotic-free medium.

To our best knowledge, no data are available on the ability of ketolides to select for resistance in H. influenzae. Our data confirm the high frequency of MIC increase caused by clarithromycin observed by others for H. influenzae, whereas differences were noted for azithromycin, probably because azithromycin selects for resistance more slowly than clarithromycin, and five passages on antibiotic-containing plates were not sufficient to determine the four-fold increase in MICs that we established to define acquisition of resistance.8

Although this study did not investigate the mechanisms or stability of mechanisms responsible for the differences in decreased susceptibility caused by the drugs under evaluation, differences in drug resistance might correlate with variation in the degree of methylation of 23S rRNA, and mutations in ribosomal proteins might confer a different degree of resistance to macrolides or ketolides, as suggested by other authors.9,10 Moreover, it may be hypothesized that mutations induced by azithromycin and clarithromycin might be more easily maintained than those caused by telithromycin.

In conclusion, in vitro exposure to telithromycin has been shown to select for mutants in respiratory pathogens less frequently than azithromycin and clarithromycin. Although the clinical relevance of this finding needs to be clarified, it may be hypothesized that telithromycin possesses a favourable resistance profile that could prevent or at least delay development of resistance occurring with other antibacterials such as macrolides.


    Footnotes
 
* Corresponding author. Tel: +39-0239042469; Fax: +39-0250319651; Email: microbio{at}unimi.it


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Baquero, F. (1999). Evolving resistance patterns of Streptococcus pneumoniae: a link with long-acting macrolide consumption? Journal of Chemotherapy 11, 35–43.[ISI][Medline]

2 . Ackermann, G. & Rodloff, A. C. (2003). Drugs of the 21st century: telithromycin (HMR 3647)—the first ketolide. Journal of Antimicrobial Chemotherapy 51, 497–511.[Abstract/Free Full Text]

3 . Bonnefoy, A., Girard, A. F., Agouridas, C. et al. (1997). Ketolides lack inducibility properties of MLSB resistance phenotype. Journal of Antimicrobial Chemotherapy 40, 85–90.[Abstract]

4 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M07-A6. NCCLS, Villanova, PA, USA.

5 . Buxbaum, A., Forsthuber, S., Graninger, W. et al. (2003). Comparative activity of telithromycin against typical community acquired-respiratory pathogens. Journal of Antimicrobial Chemotherapy 52, 371–4.[Abstract/Free Full Text]

6 . Michéa Hamzehpour, M., Kahr, A., Pechère, J. C. et al. (1994). In vitro stepwise selection of resistance to quinolones, ß-lactams and amikacin in nosocomial Gram-negative bacilli. Infection 22, Suppl. 2, S105–10.[ISI][Medline]

7 . Davies, T. A., Dewasse, B. E., Jacobs, M. R. et al. (2000). In vitro development of resistance to telithromycin (HMR 3647), four macrolides, clindamycin, and prystinamycin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 414–7.[Abstract/Free Full Text]

8 . Clark, C., Bozdogan, B., Peric, M. et al. (2002). In vitro selection of resistance in Haemophilus influenzae by amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin. Antimicrobial Agents and Chemotherapy 46, 2956–62.[Abstract/Free Full Text]

9 . Liu, M. & Douthwaite, S. (2002). Activity of the ketolide telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrobial Agents and Chemotherapy 46, 1629–33.[Abstract/Free Full Text]

10 . Tait-Kamradt, A., Davies, T., Cronan, M. et al. (2000). Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrobial Agents and Chemotherapy 44, 2118–25.[Abstract/Free Full Text]