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 Davies, T. A.
Right arrow Articles by Evangelista, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Davies, T. A.
Right arrow Articles by Evangelista, A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 2005, p. 3031-3033, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3031-3033.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Predominance of 23S rRNA Mutants among Non-Erm, Non-Mef Macrolide-Resistant Clinical Isolates of Streptococcus pneumoniae Collected in the United States in 1999-2000

Todd A. Davies,1* Karen Bush,1 Daniel Sahm,3 and Alan Evangelista2

Johnson & Johnson Pharmaceutical Research & Development, L.L.C.,1 Ortho-McNeil Pharmaceutical, Inc., Raritan, New Jersey,2 Focus Bio-Inova, Herndon, Virginia3

Received 12 January 2005/ Returned for modification 31 January 2005/ Accepted 6 March 2005


arrow
ABSTRACT
 
A total of 322 erythromycin-resistant pneumococci from TRUST 3 and TRUST 4 United States surveillance studies (1999-2000) were screened for 23S rRNA, L4, and L22 gene mutations. Nineteen isolates, two with mefA, had mutations at position 2058 or 2059 in 23S rRNA. Two had a 69GTG71-to-TPS substitution in L4; one of these also contained ermA.


arrow
TEXT
 
The most prevalent mechanisms of macrolide resistance in Streptococcus pneumoniae are mediated by mefA, a gene encoding an efflux pump specific for 14- and 15-membered macrolides, and ermB, a methylase that dimethylates A2058 in 23S rRNA, resulting in resistance to macrolides, lincosamides, and streptogramin B antibiotics. It has been shown that macrolide resistance in laboratory-generated mutants can be caused by mutations in 23S rRNA and ribosomal proteins L4 and L22 (1, 17). Recently, macrolide-resistant clinical isolates of S. pneumoniae that contain the same ribosomal mutations have been identified (3, 5, 8, 9, 12, 16).

In order to determine the specific ribosomal mutations (i.e., ribosomal protein or 23S rRNA) that exist in macrolide-resistant clinical isolates in the United States, isolates that were mefA and ermB negative were checked for mutations in the genes coding for ribosomal proteins L4 and L22 and 23S rRNA. Isolates were also checked for the presence of ermA. Specifically, from previous studies in our lab, we had in our possession 70 macrolide-resistant S. pneumoniae isolates collected in 1999 and 252 macrolide-resistant isolates collected in 2000, representing a partial collection of all the macrolide-resistant isolates from TRUST 3 and TRUST 4 surveillance studies, respectively. Most of the TRUST 3 isolates (53/70) were highly resistant to azithromycin (MIC ≥ 16 µg/ml; range, 4 to >128 µg/ml), while the 252 TRUST 4 isolates included strains with lower macrolide MICs (azithromycin MIC range, 1 to >128 µg/ml).

MICs were determined by broth microdilution using panels manufactured by Trek Diagnostic Systems (Westlake, OH) and using NCCLS methods (10). The ermB and mefA genes were detected by PCR as described by Sutcliffe et al. (14), and mutations in 23S rRNA, L4, and L22 genes were detected as described by Tait-Kamradt et al. (17). Detection of ermA by PCR was done as described by Syrogiannopoulos et al. (15). Genetic relatedness among macrolide-resistant isolates containing ribosomal mutations was determined by serotyping and pulsed-field gel electrophoresis (PFGE) as described previously (2).

The genotypes of the 70 TRUST 3 isolates and 252 TRUST 4 isolates are presented in Table 1. For the TRUST 3 isolates, ermB was the most prevalent resistance mechanism (Table 1), which can be attributed to the bias for very high levels of macrolide resistance in this isolate collection: 76% of isolates had azithromycin MICs of ≥16 µg/ml. However, mefA was the most common resistance mechanism in the TRUST 4 isolates (Table 1); these isolates had a broader range of azithromycin MICs and, hence, were more representative of the natural distribution of macrolide-resistant isolates (7). Mutations in the 23S rRNA genes were much more common than L4 mutations. Two isolates (5459 and 5486) had mefA but exhibited uncharacteristically high macrolide MICs (i.e., azithromycin MICs of >128 µg/ml) and resistance to clindamycin (Table 2). Upon further characterization, these two isolates were also found to contain 23S rRNA mutations (Table 2). In addition, one of these isolates (5486) had a mutation leading to a Glu77-to-Gly substitution in L22. While mutations in the gene encoding L22 have been shown to cause macrolide resistance in clinical isolates (5, 6, 8), this particular substitution has not been previously reported, and its role in resistance is uncertain.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Genotypes of macrolide-resistant S. pneumoniae isolates


View this table:
[in this window]
[in a new window]
  		TABLE 2. MICs and genetic analysis of macrolide-resistant S. pneumoniae isolates containing ribosomal mutations

Nineteen isolates had mutations in domain V of 23S rRNA at position 2058 or 2059 (Escherichia coli numbering), and two isolates had a three-amino-acid substitution in ribosomal protein L4 (Tables 1 and 2). All isolates with ribosomal mutations were resistant to azithromycin, clarithromycin, and erythromycin. Isolates with a A2058G mutation (three or four copies mutated) had the highest macrolide MICs, even higher than those of isolates with all four 23S rRNA gene copies with mutations at position 2059 (A to G or C) (Table 2). Seventeen of the 21 isolates with a ribosomal mutation were nonsusceptible to clindamycin (MIC, ≥0.5 µg/ml) (Table 2). While the isolate with ermA and an L4 mutation (5430) was susceptible to clindamycin by broth microdilution, a double-disk test with erythromycin and clindamycin showed this isolate to be inducibly clindamycin resistant.

A majority of isolates (14/21) with ribosomal mutations had low telithromycin MICs (≤0.06 µg/ml); however, four isolates with mutations at A2058/A2059 had elevated telithromycin MICs (0.25 to 1.0 µg/ml) (Table 2). A recent publication by Doern and Brown showed that in the PROTEKT 2000-2001 surveillance study, the telithromycin MIC90 among all isolates of S. pneumoniae was 0.5 µg/ml (4). However, among penicillin-susceptible pneumococci, the telithromycin MIC90 was 0.03 µg/ml. Three isolates with elevated telithromycin MICs in our study were penicillin-susceptible; thus, their telithromycin MICs were 8- to 32-fold higher than the reported MIC90 from the PROTEKT study (Table 2). This indicates that isolates with elevated telithromycin MICs were present before the introduction of telithromycin in the United States.

Other ribosomal-acting agents (tetracycline, linezolid, and quinupristin-dalfopristin) were tested and all isolates were susceptible except for isolate 5459, which was resistant to tetracycline (Table 2). Nine (43%) isolates were penicillin resistant (Table 2).

PFGE analyses showed that three isolates (5719, 5892, and 5948) with serotype 23F had almost identical SmaI patterns, but they did not all have the same resistance mechanisms (Table 2). Two isolates (5430 and 5501) with serotype 29 had the same PFGE pattern but different resistance mechanisms (Table 2). All other isolates had unique PFGE patterns.

The macrolide-resistant isolates with ribosomal mutations that were identified in this study had mutations primarily in domain V of 23S rRNA, and most were not clonally related. Non-Erm, non-Mef macrolide-resistant S. pneumoniae isolates from Finland were recently reported to contain mutations primarily in 23S rRNA. Some of these isolates were clonally related (12). In contrast, non-Erm, non-Mef macrolide-resistant S. pneumoniae isolates from Eastern Europe were reported to primarily have mutations in the ribosomal protein L4, and a majority of these isolates were clonally related (9, 16).

Macrolide/azalide usage continues to increase (7). Macrolide prescriptions increased 13% from 1993 to 1999, despite total antibiotic prescriptions decreasing 15% during the same time frame. The greatest increase in macrolide prescriptions was reported for children less than 5 years of age, for whom the number of prescriptions increased 320% from 1993 to 1999 (7). Twenty-nine percent (6/21) of the isolates containing ribosomal mutations in this study were isolated from children less than 5 years of age.

In summary, 21 of 322 macrolide-resistant S. pneumoniae isolates contained ribosomal mutations (23S rRNA or L4), with mutations in 23S rRNA being predominant. Two novel combinations of A2059G/mefA and L4 (69GTG71-TPS)/ermA were identified. The appearance of these mechanisms may in part be due to the continued widespread use of macrolide and azalide antibiotics. Recent TRUST 7 (2003) surveillance data showed that 27.5% of S. pneumoniae in the United States are macrolide resistant (13). How the introduction of telithromycin in the United States will affect macrolide resistance rates and the types of resistance mechanisms observed among pneumococci remains to be determined. This study provides a framework of the types of ribosomal mutations that existed among macrolide-resistant pneumococci prior to the use of telithromycin in the clinic.


arrow
ACKNOWLEDGMENTS
 
We thank Sharon Pfleger for assistance with susceptibility testing. We also thank Y. Cheung Yee and Raul Goldschmidt for their helpful discussions.

Financial support for this work was provided by Johnson & Johnson Pharmaceutical Research and Development, L.L.C., and Ortho-McNeil Pharmaceutical, Inc.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Room B225, 1000 Route 202, Raritan, NJ 08869. Phone: (908) 707-3465. Fax: (908) 707-3501. E-mail: tdavies{at}prdus.jnj.com. Back


arrow
REFERENCES
 
    1
  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. 2
  2. Davies, T. A., R. Goldschmidt, S. Pfleger, M. Loeloff, K. Bush, D. F. Sahm, and A. Evangelista. 2003. Cross-resistance, relatedness and allele analysis of fluoroquinolone-resistant United States clinical isolates of Streptococcus pneumoniae (1998-2000). J. Antimicrob. Chemother. 52:168-175.[Abstract/Free Full Text]
    3. 3
  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. 4
  4. Doern, G. V., and S. D. Brown. 2004. Antimicrobial susceptibility among community-acquired respiratory tract pathogens in the USA: data from PROTEKT US 2000-01. J. Infect. 48:56-65.[CrossRef][Medline]
    5. 5
  5. Farrell, D. J., S. Douthwaite, I. Morrissey, S. Bakker, J. Poehlsgaard, L. Jakobsen, and D. Felmingham. 2003. Macrolide resistance by ribosomal mutation in clinical isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 study. Antimicrob. Agents Chemother. 47:1777-1783.
    6. 6
  6. Farrell, D. J., I. Morrissey, S. Bakker, S. Buckridge, and D. Felmingham. 2004. In vitro activities of telithromycin, linezolid, and quinupristin-dalfopristin against Streptococcus pneumoniae with macrolide resistance due to ribosomal mutations. Antimicrob. Agents Chemother. 48:3169-3171.[Abstract/Free Full Text]
    7. 7
  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. 8
  8. Musher, D. M., M. E. Dowell, V. D. Shortridge, R. K. Flamm, J. H. Jorgensen, P. L. 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]
    9. 9
  9. 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]
    10. 10
  10. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    11. 11
  11. National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing. Twelfth informational supplement. M100-S12. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    12. 12
  12. 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]
    13. 13
  13. Sahm, D. F. 2003. Resistance issues and community-acquired respiratory infections. Clin. Cornerstone 2003(Suppl. 3):S4-S11.
    14. 14
  14. Sutcliffe, J., T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother. 40:2562-2566.[Abstract]
    15. 15
  15. 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]
    16. 16
  16. Tait-Kamradt, A., T. Davies, P. C. Appelbaum, F. Depardieu, P. Courvalin, J. Petitpas, L. Wondrack, A. Walker, M. R. Jacobs, and J. Sutcliffe. 2000. Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob. Agents Chemother. 44:3395-3401.[Abstract/Free Full Text]
    17. 17
  17. 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]

Antimicrobial Agents and Chemotherapy, July 2005, p. 3031-3033, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3031-3033.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Varaldo, P. E., Montanari, M. P., Giovanetti, E. (2009). Genetic Elements Responsible for Erythromycin Resistance in Streptococci. Antimicrob. Agents Chemother. 53: 343-353 [Full Text]  
  • Camilli, R., Del Grosso, M., Iannelli, F., Pantosti, A. (2008). New Genetic Element Carrying the Erythromycin Resistance Determinant erm(TR) in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 52: 619-625 [Abstract] [Full Text]  
  • Wierzbowski, A. K., Nichol, K., Laing, N., Hisanaga, T., Nikulin, A., Karlowsky, J. A., Hoban, D. J., Zhanel, G. G. (2007). Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian Respiratory Organism Susceptibility Study (CROSS) (1998 2004). J Antimicrob Chemother 60: 733-740 [Abstract] [Full Text]  

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 Davies, T. A.
Right arrow Articles by Evangelista, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Davies, T. A.
Right arrow Articles by Evangelista, A.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»