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Antimicrobial Agents and Chemotherapy, August 2005, p. 3520-3522, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3520-3522.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of the First Telithromycin-Resistant Streptococcus pneumoniae Isolate in Germany

Ralf René Reinert,* Mark van der Linden, and Adnan Al-Lahham

Institute for Medical Microbiology, National Reference Center for Streptococci, University Hospital, D-52057 Aachen, Germany

Received 11 April 2005/ Returned for modification 27 April 2005/ Accepted 14 May 2005


   ABSTRACT
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A total of 486 Streptococcus pneumoniae isolates were collected in 2003 and 2004 in Germany and revealed the following resistance rates: penicillin G (7.2%) and erythromycin A (18.9%). Telithromycin exhibited good in vitro activity (MIC at which 90% of the isolates tested were inhibited, 0.125 µg/ml). However, one erm(B)-positive isolate was found to be telithromycin resistant (MIC, 8 µg/ml).


   TEXT
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Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans and is responsible for both respiratory tract infections and invasive diseases (7). Resistance to macrolides has been increasingly reported for S. pneumoniae in many countries (5). Ketolides were mainly developed to overcome antibiotic resistance in pneumococci and are characterized by the lack of the L-cladinose sugar at position 3 of the erythronolide A moiety, which is replaced by a keto group. Telithromycin (HMR 3647) is the first member of this new class approved for clinical use (9). This compound was first launched in Germany in October 2001 and subsequently was introduced in Spain and Italy (February 2002), France (September 2002), and the United States (2004).

(Portions of the data were previously presented [A. Al-Lahham, N. Neuberger, S. Da Conceicao Barbosa, I. Klawitter, R. Lütticken, M. van der Linden, and R. R. Reinert, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-811, 2004].)

Between January 2003 and April 2004, consecutive clinical strains isolated from adults ≥16 years of age with pneumococcal infections were collected by 16 laboratories throughout Germany.

Confirmation of the identification of isolates was provided by positive bile solubility and by optochin susceptibility testing. MIC testing was performed using the broth microdilution method (Trek Diagnostic Systems Ltd.) as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (8). S. pneumoniae ATCC 49619 was used as a control strain. All erythromycin A-resistant strains were screened for macrolide resistance determinants using a real-time PCR procedure, as described before (11). The macrolide resistance phenotype was determined on the basis of the pattern of susceptibility to MLSB (macrolide-lincosamide-streptogramin B) antibiotics and confirmed on the basis of the double-disk (erythromycin A plus clindamycin) agar diffusion test (6). Sequencing of the 23S rRNA genes and ribosomal protein L4 and L22 genes was performed as reported earlier by Canu et al. and by Tait-Kamradt et al. (1, 15). Pneumococcal isolates were serotyped by Neufeld's Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark. Multilocus sequence typing (MLST) of the telithromycin-resistant isolate was carried out as described previously (3).

Pneumococcal strains were isolated from the following sources: blood (n = 137 [28.2%]), bronchoalveolar lavage (n = 34 [7.0%]), cerebrospinal fluid (n = 6 [1.2%]), sinus swab and puncture (n = 79 [16.3%]), sputum (n = 139 [28.6%]), tracheal secretion (n = 47 [9.7%]), and other respiratory tract sources (n = 44 [9.1%]). Antimicrobial susceptibility data of the isolates are presented in Table 1. Upon examination, 7.2% of isolates were found to be penicillin G intermediate, and only one isolate was resistant to penicillin G (MIC ≥ 2 µg/ml). Ninety-two (18.9%) and 27 (5.6%) isolates were resistant to erythromycin A and clindamycin, respectively. Only one isolate was found to be levofloxacin resistant.


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  		TABLE 1. Antimicrobial susceptibility of 486 S. pneumoniae isolates isolated in Germany between 2003 and 2004a

 
Erythromycin A-nonsusceptible isolates (n = 93) were analyzed to determine their underlying resistance determinants. Of these, 65 (69.9%) and 28 (30.1%) belonged to the mef(A) and erm(B) types of resistance, respectively. Antimicrobial susceptibility data of macrolide-resistant strains are presented in Table 2. One erm(B)-positive strain exhibited the rare iMLSB phenotype. Of note, one strain was only inhibited by 8 µg/ml of telithromycin. To our knowledge, this is the first report on a telithromycin-resistant S. pneumoniae isolated in Germany. The isolate had a cMLSB phenotype and erm(B) genotype. The strain was multiply resistant with high-level resistance to erythromycin A, clindamycin, tetracycline, and penicillin G and exhibited a pneumococcal serotype 15A. Sequencing of the 23S rRNA revealed seven mutations (pneumococcal numbering): T107G, A1513T, T1514A, A1530T, T1531A, A1535G, and A1745T. Analysis of the ribosomal protein L22 showed a wild type. In the ribosomal protein L4, a T20N alteration was found. Further analysis of the isolate by MLST classified the strain as belonging to sequence type (ST) 63.


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  		TABLE 2. Antimicrobial susceptibility of 93 erythromycin nonsusceptible S. pneumoniae isolates with different macrolide resistance determinantsa

 
The present study documented a significant increase of macrolide resistance in mainly noninvasive pneumococcal infections in adults, which parallels the increase of resistance in invasive disease in Germany (10, 17). The rate of erythromycin A resistance in pneumococci reported by this study is significantly higher than those observed earlier (9.2%) by our working group (13). Reports on telithromycin resistance in S. pneumoniae are very scarce. Farrell and Felmingham recently reported on only 10 isolates to be telithromycin resistant among a worldwide collection of 13,874 S. pneumoniae isolates isolated between 1999 and 2003. The strains isolated in France, Italy, Spain, Hungary, and Japan had telithromycin MICs of 4 to 8 µg/ml, and, as for the German isolate described in the present report, all stains showed an erm(B) genotype (4). Interestingly, further screening of the German isolate revealed additional potential resistance mechanisms, contributing to telithromycin resistance. In addition to the erm(B) resistance determinant, the strain possessed an L4 (S20N) alteration. The S20N L4 alteration has been shown to contribute to the increase of macrolide MICs in pneumococci. This observation was first made in one clinical serotype 18F pneumococcal strain isolated in 1988 in France from a blood culture (2, 4).

In a recent report by our study group on ribosomal mutations conferring resistance to macrolides in S. pneumoniae strains isolated in Germany, only two isolates were found to carry this rare L4 alteration in combination with several 23S rRNA mutations (14). One isolate in that report also showed an elevated telithromycin MIC of 0.25 µg/ml. In addition, one pneumococcal strain (Bsp11524) isolated from the eye of a 1-year-old boy in Canada that was never treated with ketolides or even macrolides showed high-level telithromycin resistance (MIC, >256 µg/ml). This strain contained a mutation in L4 (69GTG71 changed to 69TPS71) in combination with an erm(B) determinant (16). Thus, it may be assumed that the synergy of these two mechanisms leads to ketolide resistance.

In the present study, serotyping and MLST established the telithromycin-resistant isolate 1602 to belong to the serotype 15A and ST 63. Serotype 15A isolates are extremely rare among macrolide-resistant pneumococcal strains. A study on the molecular epidemiology of macrolide resistance in Europe reports on only one such isolate among a collection of 82 strains (12). Clinical characteristics of the telithromycin-resistant isolate and the isolates of the same clone as available in the MLST (www.mlst.net) database and are presented in Table 3. Isolates of ST 63 were first described in Sweden (Sweden15A-1 clone) in the early 1990s and have thereafter been isolated from meningitis and nasopharyngeal carriage in southern European countries. It is, therefore, worth mentioning that this clone may have the potential for further global spread.


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  		TABLE 3. Overview of the telithromycin-resistant strain S. pneumoniae 1602 and other multiply resistant S. pneumoniae isolates of sequence type 63e

 


   ACKNOWLEDGMENTS
 
We thank Nelli Neuberger, Sandra Da Conceicao Barbosa, and Ingrid Klawitter for excellent technical assistance. We also thank Susanne Reinert (SR Medical Communications GmbH, Frechen, Germany) for organizing and monitoring the study. We are also grateful to André Bryskier for providing the antibiotics. The authors thank the participating microbiological laboratories for providing the isolates and the responsible scientists for providing the MLST database.

The study was supported by grant RKI-415/1369235 from the German Ministry of Health (Bundesminister für Gesundheit), by a grant on community-acquired pneumonia (CAP-net) of the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung), and by a grant of Aventis, Romainville, France.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Medical Microbiology, National Reference Center for Streptococci, University Hospital, Pauwelsstrasse 30, D-52057 Aachen, Germany. Phone: 49 241 8089787. Fax: 49 241 8082483. E-mail: Reinert{at}rwth-aachen.de. Back


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Antimicrobial Agents and Chemotherapy, August 2005, p. 3520-3522, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3520-3522.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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