Comparative in vitro activity of telithromycin against macrolide-resistant and -susceptible Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae

Fiona Walsh1,*, Francis Carnegy1, Joanne Willcock2 and Sebastian Amyes1

1 Department of Medical Microbiology, Medical School, University of Edinburgh, Edinburgh EH8 9AG, UK; 2 Aventis Pharma Ltd, Kings Hill, West Malling, Kent ME19 4AH, UK

Received 31 October 2003; returned 28 December 2003; revised 2 February 2004; accepted 10 February 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The first objective was to investigate the in vitro activity of telithromycin against respiratory tract pathogens in comparison with other antimicrobial agents. The second objective was to identify the influence of the erm(B) and mef(A) genes on the susceptibility of Streptococcus pneumoniae to telithromycin.

Methods: The in vitro activity of telithromycin against S. pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, isolated from the UK and 40 macrolide-resistant S. pneumoniae from four different countries was compared with a variety of antimicrobial agents. The 140 isolates were examined for the presence of the erm(B) and mef(A) genes. The impact of 5% CO2 on susceptibility testing was also investigated.

Results: Telithromycin showed greatest activity against S. pneumoniae, but also had good activity against M. catarrhalis and H. influenzae, which was independent of their resistance profiles to other antibiotics. The MIC90 of telithromycin for S. pneumoniae was 0.12 mg/L, which was 64-fold lower than the lowest macrolide MIC; 21% of the S. pneumoniae were macrolide resistant. Thirty-eight per cent of the macrolide-resistant strains were erm(B)-positive and 62% were mef(A)-positive, but no strain contained both genes. The activity of telithromycin was similar to that of azithromycin against both M. catarrhalis and H. influenzae, Erythromycin was slightly less active: 1% and 8% of M. catarrhalis and H. influenzae, respectively, were resistant to erythromycin, but none were resistant to telithromycin. Five per cent of the S. pneumoniae strains and 4% of the H. influenzae strains changed from telithromycin susceptible to non-susceptible entirely because of the incubation conditions. The MIC50s and MIC90s of S. pneumoniae, M. catarrhalis and H. influenzae increased by one dilution when incubated in CO2.

Conclusions: Telithromycin has shown high in vitro activity against S. pneumoniae, including those strains that are macrolide susceptible and resistant as well as M. catarrhalis and H. influenzae. This study has also demonstrated that there is no cross-resistance between erythromycin and telithromycin. The impact of 5% CO2 on susceptibility testing should be investigated further before providing definite guidelines on telithromycin susceptibility testing.

Keywords: resistance, erm(B), ketolides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to antimicrobial agents is a particular problem in community-acquired respiratory tract infections (CARTIs). In some countries macrolide resistance now exceeds penicillin resistance in Streptococcus pneumoniae.1 Between 10% and 30% of Haemophilus influenzae and 85–100% of Moraxella catarrhalis worldwide are ß-lactamase producers. In the UK, erythromycin A resistance in S. pneumoniae was higher than penicillin resistance by 7.7%.1 Telithromycin is a ketolide, which represents a new generation of antimicrobial agents capable of overcoming these resistance problems. Telithromycin has activity against all three respiratory tract pathogens regardless of their resistance profiles.2,3

The purpose of this study was to evaluate the in vitro activity of telithromycin in comparison with a variety of antimicrobial agents against macrolide-susceptible and -resistant S. pneumoniae, M. catarrhalis and H. influenzae isolated over the same time period as the PROTEKT study, 1999–2001, from the UK. As the number of macrolide-resistant S. pneumoniae identified from the Edinburgh and Leeds collections was low, a cohort of macrolide-resistant isolates from Europe and America were also tested against telithromycin to investigate the activity of telithromycin against resistant isolates. This ensured that a diverse group of macrolide-resistant S. pneumoniae isolates were included in the study.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

One hundred strains each of S. pneumoniae, M. catarrhalis and H. influenzae from the UK were investigated. The S. pneumoniae were isolated from Leeds and Edinburgh, the M. catarrhalis from Edinburgh, Leeds and Wales and the H. influenzae from Edinburgh and Glasgow between 1999 and 2001. Forty macrolide-resistant S. pneumoniae isolates were donated from the USA, Canada, Belgium and Italy. These isolates were donated by Dr Zhong (Abbott Laboratories, USA), Dr de Azavedo (University of Toronto, Canada), Dr Lagrou (Rega Institute for Medical Research, Belgium) and Dr Marchese (University of Genova, Italy), respectively. The control strains consisted of S. pneumoniae NCTC 13593, Staphylococcus aureus NCTC 6571, H. influenzae NCTC 11931 and a laboratory reference strain of M. catarrhalis.

Antimicrobial agents

The antimicrobial agents were stored and prepared according to the manufacturers’ guidelines. Telithromycin (Aventis Pharma Ltd), erythromycin (David Bull Laboratories, Warwick, UK), azithromycin (Pfizer Ltd), clindamycin (Sigma), moxifloxacin (Bayer), gemifloxacin (SmithKline Beecham Pharmaceuticals) and linezolid (Pharmacia and Upjohn) were tested in vitro.

Minimum inhibitory concentrations

The MICs were determined on Columbia agar base supplemented with 5% defibrinated horse blood for S. pneumoniae and M. catarrhalis and on chocolate Columbia agar plates for H. influenzae according to the BSAC guidelines for susceptibility testing.4 The MICs were determined by the standard agar doubling dilution method. The MIC plates containing telithromycin were incubated in both air and 5% CO2, at 37°C. All other plates were incubated in air at 37°C. The MIC tests were repeated at least once for each strain.

The antimicrobial breakpoints used were those from the BSAC guidelines except for telithromycin for which the NCCLS tentative breakpoints were used.5,6 Resistance to the antimicrobial agents was assigned at the following MIC values: telithromycin >=4 mg/L for S. pneumoniae and >=16 mg/L for H. influenzae, erythromycin >=1 mg/L, azithromycin >=2 mg/L and clindamycin >=1 mg/L.

Characterization of macrolide resistance mechanism

S. pneumoniae strains with an erythromycin MIC >= 1 mg/L or an azithromycin MIC >= 2 mg/L isolated from the UK and the 40 donated isolates were screened for the presence of the erm(B) and mef(A) genes. S. pneumoniae strains were emulsified in 200 µL of MilliQ water and boiled for 10 min in order to extract the total DNA. The supernatant was used as the DNA template in PCR experiments. The PCR conditions and primers for the detection of erm(B) and mef(A) genes are those described previously by Sutcliffe et al.7 and Tait-Kamradt et al.8 The strains were tested for both genes at the same time. The erm(B) and mef(A) primers were added to the same reaction mixture with a MgCl2 concentration of 4 mM.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility profiles

Telithromycin had the second lowest MIC90 of all the antimicrobial agents tested against S. pneumoniae as shown in Table 1. The telithromycin MIC90 was 64-fold lower than the lowest macrolide MIC90. While the MIC90 of clindamycin was the same as that of telithromycin, the upper limit of the range was substantially higher. Telithromycin activity against the 40 macrolide-resistant S. pneumoniae was the same as that against the 100 UK isolates with varying macrolide susceptibilities. The range endpoint (1 mg/L) was lower than that for the UK isolates (2 mg/L) and the MIC90s (0.25 mg/L) were identical. Thus telithromycin activity was not affected by the macrolide resistance mechanisms of these strains. Gemifloxacin and moxifloxacin both had high activity against S. pneumoniae with their highest MICs at 0.12 and 0.5 mg/L, respectively. Linezolid did not perform as well as the other antimicrobial agents. The fluoroquinolones had the greatest activity of the antimicrobial agents tested against M. catarrhalis. Their MIC90s ranged from 0.016 to 0.06 mg/L. The MIC90s of the macrolides were 0.12 and 0.25 mg/L. There was little difference between the performance of telithromycin and the macrolides. Clindamycin and linezolid both had low activity against M. catarrhalis in vitro with MIC90s of 4 mg/L. Telithromycin had relatively good activity against H. influenzae with an MIC90 of 2 mg/L. It had lower MIC50 and MIC90 values than erythromycin by two dilutions. For H. influenzae, clindamycin and linezolid had the same MIC90s of 16 mg/L. Once again the fluoroquinolones performed with the highest activity and low MIC90s of 0.004–0.016 mg/L.


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Table 1. Antimicrobial activities against S. pneumoniae, M. catarrhalis and H. influenzae
 
Correlation between telithromycin and erythromycin

The correlation between telithromycin MIC and that of erythromycin and clindamycin for S. pneumoniae are detailed in Table 2. These results indicated that the five isolates with the highest telithromycin MICs were erythromycin resistant but clindamycin susceptible. Eight of the 12 isolates with a telithromycin MIC of 0.12 mg/L were erythromycin resistant, and three of these were also clindamycin resistant. The S. pneumoniae isolates resistant to clindamycin had telithromycin MICs of 0.032–0.12 mg/L and were also erythromycin resistant.


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Table 2. Comparison of telithromycin susceptibility and erythromycin and clindamycin resistance profiles of S. pneumoniae
 
Effect of CO2 incubation

When the MIC plates containing telithromycin were incubated in 5% CO2 (Table 1) the range and MIC90 for S. pneumoniae increased by one dilution and the MIC50 increased by two dilutions. The S. pneumoniae range endpoint increased from 1 to 2 mg/L when incubated in CO2. With a telithromycin MIC of 2 mg/L a S. pneumoniae strain is categorized as intermediate using the BSAC guidelines and resistant using the French guidelines. Five per cent of S. pneumoniae were telithromycin resistant and 2% or 5% had intermediate resistance using the French guidelines and BSAC guidelines, respectively, when measured in CO2. With regard to M. catarrhalis and H. influenzae the MIC50 and MIC90 both increased by one dilution when incubated in CO2. The approved telithromycin breakpoint for H. influenzae is 4 mg/L for susceptible, 8 mg/L for intermediate and >=16 mg/L for resistant. Strains with an MIC of 4 mg/L in air were categorized as susceptible but with CO2 incubation showed an MIC of 8 mg/L and were categorized as intermediate.

Resistance levels

Telithromycin had the lowest resistance levels, considerably lower than either the macrolides or the lincosamide. There was no resistance to telithromycin in either S. pneumoniae, M. catarrhalis or H. influenzae using the NCCLS breakpoints (Table 3). Using the guidelines suggested by Soussy et al.,9 i.e. telithromycin susceptibility <0.5 mg/L and resistance >=2 mg/L, telithromycin intermediate resistance of 1 mg/L for Gram-positive bacteria, the resistance levels for S. pneumoniae increased slightly. One per cent of S. pneumoniae isolates had telithromycin intermediate resistance. In comparison, 21% of the S. pneumoniae population was erythromycin resistant and 6% were resistant to clindamycin. In M. catarrhalis the entire population were found to be clindamycin resistant while one strain was also erythromycin resistant. No resistance to telithromycin or azithromycin was observed. Erythromycin resistance was at 8% for H. influenzae, whereas azithromycin resistance was at 1%.


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Table 3. Resistance profiles of S. pneumoniae, M. catarrhalis and H. influenzae
 
Mechanisms of macrolide resistance

The 21 macrolide-resistant S. pneumoniae strains consisted of eight erm(B)-positive and 13 mef(A)-positive strains. There were no strains containing both genes. All the strains except two Edinburgh isolates had matching phenotype and genotype, i.e. erm(B)-positive strains were macrolide and clindamycin resistant and mef(A)-positive strains were resistant only to the macrolides. The Edinburgh erm(B)-positive strains had clindamycin MICs of 0.12 and 0.25 mg/L. The 40 donated strains comprised 29 erm(B)-positive and 11 mef(A)-positive strains. No strains contained both genes. Within this group four strains were also erm(B)-positive but clindamycin susceptible with MICs of 0.12 or 0.25 mg/L.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility testing of respiratory tract pathogens is often performed in a CO2 environment to ensure that the bacteria grow to the required concentration. In a CO2 environment the pH of the test medium decreases. Macrolide activity is adversely affected by this pH decrease. In order to investigate whether this was also true for the ketolide telithromycin, susceptibility testing was also carried out in 5% CO2. This study has shown that the MIC of telithromycin increased when incubated in 5% CO2 in comparison with air incubation. Five per cent of the S. pneumoniae strains and 4% of the H. influenzae strains changed from telithromycin susceptible to non-susceptible entirely because of the incubation conditions. The MIC50s and MIC90s of S. pneumoniae, M. catarrhalis and H. influenzae increased by one dilution when incubated in CO2. These results indicated that the pH of the medium also caused an increase in telithromycin MIC. An increase in the telithromycin mode MIC and MIC range was also detected by Bemer-Melchoir et al.10 when the susceptibility of Streptococcus pyogenes was tested in 5–6% CO2. However, unlike the results described by Bemer-Melchoir et al., which suggested that the MIC of telithromycin in CO2 increased only for erythromycin-resistant isolates, no such correlation was identified with S. pneumoniae, M. catarrhalis or H. influenzae. In this and previous studies macrolide resistance has been associated mainly with S. pneumoniae rather than M. catarrhalis or H. influenzae.1

The French guidelines recommend testing telithromycin activity against S. pneumoniae in air as well as CO2 in order to confirm resistance for clinical characterization. However, no such recommendations were presented for H. influenzae. The results of this study suggest that telithromycin susceptibility should be tested in air for all three organisms. Therefore, the French committee need to investigate the effect of incubation in CO2 for H. influenzae also. The latest BSAC guidelines recommend that S. pneumoniae and H. influenzae are incubated in CO2 and M. catarrhalis is incubated in air for susceptibility testing. Under these conditions the tentative susceptibility to telithromycin point for the three organisms is <0.5 mg/L. As incubation in CO2 has affected each of the three organisms tested in this study it would be appropriate for the BSAC to investigate this further with these and other organisms and larger populations before providing definite guidelines on telithromycin susceptibility testing and breakpoints.11

In the Alexander Project, erythromycin resistance in S. pneumoniae from the UK was at 13.6% in 1996 and 7.2% in 1997.12 However, this study has shown that the resistance level in Edinburgh and Leeds for the same period was 21% in S. pneumoniae. The percentage of H. influenzae resistant to erythromycin is 8%; however, none are telithromycin resistant and almost all are azithromycin susceptible.

The results of the correlation between telithromycin, erythromycin and clindamycin identified a pattern similar to that described previously by Johnson et al.13 All the erythromycin- and clindamycin-resistant isolates were susceptible to telithromycin. The telithromycin MICs for the isolates that were erythromycin resistant alone or in combination with clindamycin resistance were higher than those for the susceptible isolates. The S. pneumoniae with an erythromycin-resistant and clindamycin-susceptible phenotype were less susceptible to telithromycin than those resistant to both. The isolates with the highest telithromycin MICs were erythromycin resistant and clindamycin susceptible. Therefore, in agreement with Johnson et al.13 telithromycin was less affected by the erm(B)-positive than by the mef(A)-positive isolates. Thus, while telithromycin can overcome both resistance mechanisms as all isolates were telithromycin susceptible, the macrolide efflux system and to a lesser extent the target methylation do reduce telithromycin activity.

Telithromycin has previously been shown to have activity against macrolide-resistant S. pneumoniae. This finding has been borne out in these results as it was tested against 61 macrolide-resistant S. pneumoniae isolates. Telithromycin had consistently lower MICs than erythromycin for all the bacteria tested. No resistance to telithromycin was found in S. pneumoniae, M. catarrhalis or H. influenzae. The 21 macrolide-resistant S. pneumoniae isolates from the UK comprised erm(B)- and mef(A)-positive isolates in a ratio of 1:2. Usually in Europe the erm(B) gene predominates and the converse is found in the USA. This study contradicts this finding in so far as there was a greater number of mef(A)-positive UK isolates than erm(B)-positive isolates. Isolates that were erythromycin and clindamycin resistant have been attributed a constitutive macrolide resistance phenotype and those that were erythromycin resistant and clindamycin susceptible an inducible macrolide resistance phenotype.14 Therefore, the results of this study indicate that the constitutive resistance phenotype predominates in the isolates investigated in this study. Thus telithromycin has activity against inducible macrolide-resistant S. pneumoniae and also against the inducible macrolide-resistant S. pneumoniae investigated in this study. The high overall activity of telithromycin against the three respiratory pathogens and almost no resistance indicates that telithromycin has good potential for the treatment of RTIs, including those caused by macrolide-resistant strains.


    Acknowledgements
 
This study was supported by a grant from Aventis Pharma Ltd.


    Footnotes
 
* Corresponding author. Tel: +44-131-650-8270; Fax: +44-131-651-1385; E-mail: fwalsh{at}staffmail.ed.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Felmingham, D. (2002). Evolving resistance patterns in community-acquired respiratory tract pathogens: first results from the PROTEKT global surveillance study. Journal of Infection 44, 3–10.[ISI][Medline]

2 . Barry, A. L., Fuchs, P. C. & Brown, S. D. (1998). In vitro activities of the ketolide HMR 3647 against recent gram-positive clinical isolates and Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 42, 2138–40.[Abstract/Free Full Text]

3 . Wootton, M., Bowker, K. E., Janowska, A. et al. (1999). In vitro activity of HMR 3647 against Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and ß-haemolytic streptococci. Journal of Antimicrobial Chemotherapy 44, 445–53.[Abstract/Free Full Text]

4 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

5 . MacGowan, A. P. & Wise, R. (2001). Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. Journal of Antimicrobial Chemotherapy 48 Suppl. S1, 17–28.

6 . Bearden, B. T., Neuhauser, M. M. & Garey, K. M. (2001). An oral ketolide for respiratory infections. Pharmacotherapy 21, 1204–22.[ISI][Medline]

7 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. et al. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

8 . Tait-Kamradt, A., Clancy, J., Cronan, M. et al. (1997). mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 2251–5.[Abstract]

9 . Members of the SFM Antibiogram Committee. (2003). Comité de l’Antibiogramme de la Societé Française de Microbiologie Report 2003. International Journal of Antimicrobial Agents. 21 364–91.[CrossRef][ISI][Medline]

10 . Bemer-Melchoir, P., Juvin, M. E., Tassin, S. et al. (2000). In vitro activity of the new ketolide telithromycin compared with those of macrolides against Streptococcus pyogenes: influences of resistance mechanisms and methodological factors. Antimicrobial Agents and Chemotherapy 44, 2999–3002.[Abstract/Free Full Text]

11 . BSAC Working Party. (2003). BSAC Disc Diffusion Method for Antimicrobial Susceptibility Testing Version 2.1.4 [Online.] http://www.bsac.org.uk (5 March 2004, date last accessed).

12 . Felmingham, D., Grüneberg, R. N. & the Alexander Project Group. (2000). The Alexander Project 1996–1997: latest susceptibility data from this international study of bacterial pathogens from community-acquired lower respiratory tract infections. Journal of Antimicrobial Chemotherapy 45, 191–203.[Abstract/Free Full Text]

13 . Johnson, A. P., Henwood, C. J., Tysall, L. et al. (2001). Activity of the ketolide telithromycin (HMR-3647) against erythromycin-susceptible and -resistant pneumococci isolated in the UK. International Journal of Antimicrobial Agents 18, 73–6.[CrossRef][ISI][Medline]

14 . Reinert, R. R., Wild, A., Appelbaum, A. et al. (2003). Ribosomal mutations conferring resistance to macrolides in Streptococcus pneumoniae clinical strains isolated in Germany. Antimicrobial Agents and Chemotherapy 47, 2319–22.[Abstract/Free Full Text]