AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reinert, R. R.
Right arrow Articles by Al-Lahham, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reinert, R. R.
Right arrow Articles by Al-Lahham, A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 2003, p. 2319-2322, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2319-2322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Ribosomal Mutations Conferring Resistance to Macrolides in Streptococcus pneumoniae Clinical Strains Isolated in Germany

Ralf René Reinert,1* Angela Wild,1 Peter Appelbaum,2 Rudolf Lütticken,1 Murat Yücel Cil,1 and Adnan Al-Lahham1

Institute of Medical Microbiology, National Reference Center for Streptococci, Aachen, Germany,1 Hershey Medical Center, Hershey, Pennsylvania2

Received 19 December 2002/ Returned for modification 12 March 2003/ Accepted 7 April 2003


   ABSTRACT
Top
 Abstract
Text
 References
 
Among a collection of 4,281 pneumococcal isolates, 7 strains isolated in Germany had an unusual macrolide resistance phenotype. The isolates were found to have multiple mutations in the 23S rRNA and alterations in the L4 ribosomal protein. One strain had an amino acid alteration in the L22 ribosomal protein.


   TEXT
Top
 Abstract
 Text
References
 
Resistance to macrolides is increasingly being reported among clinical isolates of Streptococcus pneumoniae worldwide (7). In Germany a dramatic increase in the rate of macrolide resistance among pneumococcal strains isolated from patients with invasive disease was observed only recently (17, 18).

The most prevalent mechanisms of macrolide resistance in S. pneumoniae are mediated by mef(A), a gene encoding an efflux pump, and erm(B), a 23S rRNA methylase that dimethylates the adenine at position 2058, resulting in macrolide-lincosamide-streptogramin B (MLSB) resistance (19). MLSB resistance can be expressed either constitutively (cMLSB phenotype) or inducibly (iMLSB phenotype) (12). In addition, mutations in 23S rRNA and ribosomal proteins L4 and L22 have been shown to account for resistance in pneumococci (8, 9, 11, 13, 14, 22). In rare cases the presence of an erm(A) gene in pneumococci has been reported (20).

Three surveillance studies on antibiotic resistance screened for strains with unusual macrolide resistance genotypes or phenotypes. Strains were included in the present investigation if they were macrolide resistant and showed the erm(B)-negative and mef(A)-negative genotype in repeated PCR assays (five strains) or a discrepancy between the macrolide resistance genotype and phenotype (two strains). The characteristics of the studies are presented in Table 1 (5, 17, 25). Each isolate was confirmed as S. pneumoniae by optochin sensitivity and bile solubility testing. MICs were determined by the broth microdilution method, as described by the NCCLS (15). Determination of macrolide-resistant phenotypes was performed as described previously (12). The primers described by Trieu-Cuot et al. (23) and Tait-Kamradt et al. (21) were chosen for the detection of erm(B) and mef(A). Sequencing of the 23S rRNA genes and ribosomal protein L4 and L22 genes was performed as reported earlier by Canu et al. (1) and Tait-Kamradt et al. (22). The number of mutated copies of the 23S rRNA was determined as described by Canu et al. (1). Strains were serotyped by Neufeld's Quellung reaction, and the genetic relatedness of strains was analyzed by multilocus sequence typing (MLST) (4).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Macrolide resistance and resistance genotypes of 4,281 pneumococcal strains documented by nationwide studies in Germany, 1992 to 2002a

 
The genotypes and phenotypes of these strains are presented in Table 2; and their serotypes, mutations in 23S rRNA, and changes in ribosomal proteins L4 and L22 are presented in Table 3.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Patient demographic data and MICs for seven macrolide-resistant strains isolated in Germany, 1992 to 2000a

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Mutations in 23S rRNA and changes in ribosomal proteins L4 and L22 in macrolide-resistant pneumococcal strains

 
Strain NRZ 288 exhibited three point mutations in the 23S rRNA gene and two amino acid alterations in ribosomal protein L4. The strain showed a cMLSB phenotype, high levels of resistance to all macrolides, and a relatively low level of resistance to clindamycin compared to the resistance of the erm(B) strains with constitutive resistance (MIC for NRZ 288, 8 µg/ml). Strain NRZ 796 also showed three mutations in the 23S rRNA in combination with a change in protein L4 (S20N). Of note, this strain showed an inducible phenotype, and another efflux mechanism like the new efflux mechanism recently described in Streptococcus pyogenes may be present (6). Strain PS 2938 showed two mutations (G211A and T389C) in the 23S rRNA and one alteration (R95H) in ribosomal protein L4. This strain exhibited the cMLSB phenotype and high-level resistance to all macrolides and clindamycin. Strain PS 2909 showed a single mutation (T389C) in the 23S rRNA gene. Among the strains in the collection, this was the only isolate showing a change (C117T) in protein L22. Multiple changes were found in ribosomal protein L4. Strain PW 555 showed mutations only downward to the 3' end of the 23S rRNA. Strain NRZ 462 expressed the erm(B) macrolide resistance gene in repeated PCR assays and showed high-level resistance to all macrolides. Interestingly, this strain showed the iMLSB phenotype. Sequencing of the 23S rRNA gene of strain NRZ 462 showed four mutations (A138G, A376T, T389C, and G2135T). Strain NRZ 810 showed three mutations (A138G, T139C, and T389C) in the 23S rRNA gene. This strain was mef(A) positive but showed high-level resistance to both macrolides and clindamycin (MICs, ≥32 µg/ml).

All strains were isolated from different geographic areas in Germany and from individuals in various age groups. The strains were genetically unrelated and, with one exception, represented different serotypes (Table 3). MLST demonstrated that all strains belonged to different sequence types. Interestingly, three of the seven strains belonged to new multilocus sequence types not reported in the MLST database to date (Table 3).

Most information available today is based on in vitro selection studies showing that certain structures involving domains II and V of 23S rRNA participate in the binding of macrolides (1-3). In clinical isolates most of the point mutations were identical to those found in in vitro selection studies, but new mutations were also observed (11). The A2058G and A2058U substitutions confer the highest level of MLSB resistance, with the MICs of erythromycin and related macrolides being between 32 and >200 µg/ml (1, 14, 22, 24).

A clinical isolate that had an A2062C mutation and that exhibited high-level resistance to spiramycin and streptogramin B has recently been described by Depardieu and Courvalin (3). Other mutations reported to date include C2610 and C2611 (which confer low-level macrolide resistance) (1, 22).

Mutations in the L4 protein occur in a region of 32 amino acids and interfere with the binding of the protein to rRNA (8, 14, 16). A 3-amino-acid alteration of GTG to TPS at position 69 was also seen in strain PS 2909 in the present investigation.

Two isolates in the present study showed the S20N alteration in the L4 protein. The latter has been described in only one other clinical serotype 18F strain, which was isolated in 1988 in France and which exhibited the S20N alteration in the L4 protein in combination with an A2062C mutation in the 23S rRNA. However, the investigators demonstrated that the S20N mutation was not involved in the expression of macrolide resistance in this strain (3).

In the present study only one clinical isolate (PS 2909) showed a mutation in the L22 protein. Mutations in the L22 protein are usually located in a ß-hairpin extension at the C terminus of the protein (1, 11). A prvevious study (13) reported the emergence of a mutant with an L22 protein mutation during treatment with azithromycin in a patient with fatal pneumococcal pneumonia.

In summary, the seven macrolide-resistant strains isolated in Germany showed some of the well-known mutations in the 23S rRNA and L4 ribosomal protein, but they also exhibited new mutations and new combinations of L4 and 23S rRNA mutations. A new alteration in the L22 protein which has not been reported before is described. Two strains showed a genotype that did not match the phenotype, which has been described in pneumococci before (10). In addition, the relevance of the newly described mutations for the development of macrolide resistance in pneumococci needs to be confirmed by future experiments.


   ACKNOWLEDGMENTS
 
We thank Nelli Neuberger and Claudia Cremer for excellent technical assistance. We thank André Bryskier for providing the antibiotics and Susan Griesbach for carefully reading the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology, National Reference Center for Streptococci, University of Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. Phone: 49 241 80 89 787. Fax: 49 241 8082 483. E-mail: reinert{at}rwth-aachen.de. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Canu, A., B. Malbruny, M. Coquemont, T. A. Davies, P. C. Appelbaum, and R. Leclercq. 2002. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 46:125-131.[Abstract/Free Full Text]
  2. Davies, T. A., B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum. 2000. In vitro development of resistance to telithromycin (HMR 3647), four macrolides, clindamycin, and pristinamycin in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:414-417.[Abstract/Free Full Text]
  3. Depardieu, F., and P. Courvalin. 2001. Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 45:319-323.[Abstract/Free Full Text]
  4. Enright, M. C., and B. G. Spratt. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049-3060.[Abstract]
  5. Felmingham, D., R. R. Reinert, Y. Hirakata, and A. Rodloff. 2002. Increasing prevalence of antimicrobial resistance among isolates of Streptococcus pneumoniae from the PROTEKT surveillance study, and comparative in vitro activity of the ketolide, telithromycin. J. Antimicrob. Chemother. 50(Suppl. S1):25-37.[Abstract]
  6. Giovanetti, E., A. Brenciani, R. Burioni, and P. E. Varaldo. 2002. A novel efflux system in inducibly erythromycin-resistant strains of Streptococcus pyogenes. Antimicrob. Agents Chemother. 46:3750-3755.[Abstract/Free Full Text]
  7. Hyde, T. B., K. Gay, D. S. Stephens, D. J. Vugia, M. Pass, S. Johnson, N. L. Barrett, W. Schaffner, P. R. Cieslak, P. S. Maupin, E. R. Zell, J. H. Jorgensen, R. R. Facklam, and C. G. Whitney. 2001. Macrolide resistance among invasive Streptococcus pneumoniae isolates. JAMA 286:1857-1862.[Abstract/Free Full Text]
  8. Kozlov, R. S., T. M. Bogdanovitch, P. C. Appelbaum, L. Ednie, L. S. Stratchounski, M. R. Jacobs, and B. Bozdogan. 2002. Antistreptococcal activity of telithromycin compared with seven other drugs in relation to macrolide resistance mechanisms in Russia. Antimicrob. Agents Chemother. 46:2963-2968.[Abstract/Free Full Text]
  9. Leclercq, R. 2002. Will resistance to ketolides develop in Streptococcus pneumoniae? J. Infect. 44(Suppl. A):11-16.
  10. Leclercq, R., and P. Courvalin. 1993. Mechanisms of resistance to macrolides, p. 125-141. In A. Bryskier, J. Butzler, H. C. Neu, and P. M. Tulkens (ed.), Macrolides—chemistry, pharmacology, and clinical uses. Arnette Blackwell, Paris, France.
  11. Leclercq, R., and P. Courvalin. 2002. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 46:2727-2734.[Free Full Text]
  12. Montanari, M. P., M. Mingoia, E. Giovanetti, and P. E. Varaldo. 2001. Differentiation of resistance phenotypes among erythromycin-resistant pneumococci. J. Clin. Microbiol. 39:1311-1315.[Abstract/Free Full Text]
  13. Musher, D. M., M. E. Dowell, V. D. Shortridge, R. K. Flamm, J. H. Jorgensen, P. Le Magueres, and K. L. Krause. 2002. Emergence of macrolide resistance during treatment of pneumococcal pneumonia. N. Engl. J. Med. 346:630-631.[Free Full Text]
  14. Nagai, K., P. C. Appelbaum, T. A. Davies, L. M. Kelly, D. B. Hoellman, A. T. Andrasevic, L. Drukalska, W. Hryniewicz, M. R. Jacobs, J. Kolman, J. Miciuleviciene, M. Pana, L. Setchanova, M. K. Thege, H. Hupkova, J. Trupl, and P. Urbaskova. 2002. Susceptibilities to telithromycin and six other agents and prevalence of macrolide resistance due to L4 ribosomal protein mutation among 992 pneumococci from 10 central and Eastern European countries. Antimicrob. Agents Chemother. 46:371-377.[Abstract/Free Full Text]
  15. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically (M7-A4). Approved standard (M100-S8), 4th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  16. Pihlajamaki, M., J. Kataja, H. Seppala, J. Elliot, M. Leinonen, P. Huovinen, and J. Jalava. 2002. Ribosomal mutations in Streptococcus pneumoniae clinical isolates. Antimicrob. Agents Chemother. 46:654-658.[Abstract/Free Full Text]
  17. Reinert, R. R., A. Al-Lahham, M. Lemperle, C. Tenholte, C. Briefs, S. Haupts, H. H. Gerards, and R. Lütticken. 2002. Emergence of macrolide and penicillin resistance among invasive pneumococcal isolates in Germany. J. Antimicrob. Chemother. 49:61-68.[Abstract/Free Full Text]
  18. Reinert, R. R., R. Lütticken, A. Bryskier, and A. Al-Lahham. 2003. Macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes in the pediatric population in Germany during 2000-2001. Antimicrob. Agents Chemother. 47:489-493.[Abstract/Free Full Text]
  19. Sutcliffe, J., A. Tait Kamradt, and L. Wondrack. 1996. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrob. Agents Chemother. 40:1817-1824.[Abstract]
  20. Syrogiannopoulos, G. A., I. N. Grivea, A. Tait-Kamradt, G. D. Katopodis, N. G. Beratis, J. Sutcliffe, P. C. Appelbaum, and T. A. Davies. 2001. Identification of an erm(A) erythromycin resistance methylase gene in Streptococcus pneumoniae isolated in Greece. Antimicrob. Agents Chemother. 45:342-344.[Abstract/Free Full Text]
  21. Tait-Kamradt, A., J. Clancy, M. Cronan, F. Dib-Hajj, L. Wondrack, W. Yuan, and J. Sutcliffe. 1997. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 41:2251-2255.[Abstract]
  22. Tait-Kamradt, A., T. Davies, M. Cronan, M. R. Jacobs, P. C. Appelbaum, and J. Sutcliffe. 2000. Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrob. Agents Chemother. 44:2118-2125.[Abstract/Free Full Text]
  23. Trieu-Cuot, P., C. Poyart-Salmeron, C. Carlier, and P. Courvalin. 1990. Nucleotide sequence of the erythromycin resistance gene of the conjugative transposon Tn1545. Nucleic Acids Res. 18:3660.[Free Full Text]
  24. Vester, B., and S. Douthwaite. 2001. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob. Agents Chemother. 45:1-12.[Free Full Text]
  25. von Kries, R., A. Siedler, H. J. Schmitt, and R. R. Reinert. 2000. Proportion of invasive pneumococcal infections in German children preventable by pneumococcal conjugate vaccines. Clin. Infect. Dis. 31:482-487.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, July 2003, p. 2319-2322, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2319-2322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reinert, R. R.
Right arrow Articles by Al-Lahham, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reinert, R. R.
Right arrow Articles by Al-Lahham, A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS