ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2006, p. 3897–3900 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.00057-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 11
Telithromycin-Nonsusceptible Clinical Isolates of Streptococcus pneumoniae from Europe Adnan Al-Lahham,1† Peter C. Appelbaum,2 Mark van der Linden,1 and Ralf Rene´ Reinert1* Institute of Medical Microbiology, National Reference Center for Streptococci, University Hospital, Aachen, Germany,1 and Hershey Medical Center, Hershey, Pennsylvania2 Received 13 January 2006/Returned for modification 13 March 2006/Accepted 30 July 2006
Telithromycin-nonsusceptible pneumococcal clinical isolates (n ⴝ 17) were analyzed for their antimicrobial susceptibility, macrolide resistance mechanisms, and genetic relatedness. All strains showed the erm(B) genotype and showed a wide range of combinations of macrolide resistance mechanisms. The predominant clone (n ⴝ 7) was serotype 14, sequence type 143. Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans (14). The worldwide increase in antibiotic resistance in this species has become a serious problem within the last 20 years (2). Macrolide resistance in S. pneumoniae is usually caused by methylation of A2058 of the 23S rRNA mediated by the erm(B) gene or by an efflux mechanism mediated by the mef gene (13). In addition, other mechanisms of macrolide resistance have been described (4, 20, 21). Ketolides form a new class of semisynthetic agents derived from erythromycin A designed to overcome erythromycin A resistance in S. pneumoniae. Ketolide compounds inhibit bacterial protein synthesis by interacting with the peptidyltransferase site of the 50S ribosomal subunit and interact closely with domains II, at A752, and V, at A2058 and A2059, of the 23S rRNA (1). Ketolides show good activity against gram-positive bacteria responsible for respiratory tract infections, including penicillin G- and erythromycin A-resistant S. pneumoniae (11). The ketolide-resistant pneumococci described usually show the erm(B) genotype, and some, but not all, strains show additional alterations in the L4 ribosomal protein (1, 16). The scope of the present paper was to investigate the occurrence of these mechanisms of resistance in a collection of 17 telithromycin-nonsusceptible pneumococcal isolates from Europe. (This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, 16 to 19 December 2005, Washington, D.C. [1a]). MIC testing was performed using the broth microdilution method recommended by the Clinical Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) (5). PCR of macrolide resistance genes was performed as described previously (17). Sequencing of the 23S rRNA and the genes encoding the ribosomal proteins L4 (rplD) and L22 (rplV) was performed as reported earlier by Tait-Kamradt et al. (4, 21) and Walsh et al. (22). The erm(B) gene and the erm(B) promoter region were amplified by PCR
using the primers published by Walsh et al. (22). Pneumococcal strains were serotyped by Neufeld’s Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark. Multilocus sequence typing (MLST) was carried out as described by Enright and Spratt (8). Phylogenetic analysis was performed as described before (18). Nucleotide sequences of the upstream regions of the erm gene were aligned using the program ClustalW. Sequence types (STs) of the telithromycin-nonsusceptible isolates were compared with those of all isolates available in the MLST database. Demographic data and data on antibiotic resistance are presented in Table 1. Isolates were collected between 2001 and 2005 from patients with invasive and noninvasive pneumococcal disease, mostly in France (10 out of 17 strains). Telithromycin MICs ranged between 2 and 8 g/ml. All strains were multiply resistant and besides being telithromycin nonsusceptible showed reduced susceptibility to -lactams and resistance to macrolides, lincosamides, and tetracycline. One strain additionally showed high-level fluoroquinolone resistance. Genotypic and phenotypic characteristics and results from MLST are presented in Table 2. The predominant serotype was type 14 (10 of 17 isolates). The predominant clone was ST 143 (7 of 17 isolates), which was found in France (n ⫽ 6) and Germany (n ⫽ 1). Telithromycin resistance has also spread to the Spain23F-1 clone (ST 81; n ⫽ 1) and its 19A variant. All strains showed the erm(B) genotype and the cMLSB phenotype and were negative for the mef gene. The isolates had multiple alterations in 23S rRNA. Analysis of the genes encoding ribosomal protein L22 showed a wild type for all isolates. Of note, in nine isolates an S20N alteration in ribosomal protein L4 was observed. Isolates belonging to one clone showed identical or quite similar resistance mechanisms. Of note, three clones were described for the first time in the present investigation (strain PW 778, ST 1569; PW 735, ST 1558; and PW 868, ST 2010) (Table 3). The DNA sequences of the erm(B) upstream region of the telithromycin-nonsusceptible clinical isolates were compared to the upstream sequence region of erm(B) found in Tn1545 (accession no. X52632) (15), erm(B) (accession no. AJ972605) (7), pAM77 (accession no. K00551) (12), and Tn917-like LP-1 (accession no. AB111455) (15) (Fig. 1). In the region upstream of the erm(B) start codon, mutations were found at positions
* Corresponding author. Mailing address: Institute for Medical Microbiology, National Reference Center for Streptococci, University Hospital (RWTH), Pauwelsstr. 30, Aachen, Germany. Phone: 49 241 8089946. Fax: 49 241 8082483. E-mail:
[email protected]. † Present address: German Jordanian University, School of Applied Medical Sciences, Amman, Jordan. 3897
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TABLE 1. Characteristics, MICs, and resistance phenotypes of 17 telithromycin-nonsusceptible S. pneumoniae strains from Europe Strain
PW 778 PW 521 PW 868 PW 899 PW 1760 PW 1765 PW 1791 PW 1840 PW 1981 PW 1976 PW 1996 PN 302 PS 5646 PW 712 PW 715 PW 735 PW 1420
Country
Belgium France France France France France France France France France France Germany Germany Spain Spain Spain Switzerland
Source
Sputum BALc BAL BAL Sinus BAL Sputum Blood Sputum Sputum Blood Blood BAL Sputum Sputum Sputum BAL
MIC (g/ml) of a:
Yr of isolation
Patient age (yr)
TEL
PEN
AMX
CTX
CLR
CLI
TET
LVX
SXT
2000 2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2003 2005 2001 2001 2001 2002
67 44 59 64 31 57 53 59 69 ND 81 34 58 56 70 75 30
8 8 2 4 4 8 8 4 4 2 2 8 8 4 2 4 8
2 4 4 4 2 2 1 2 2 2 4 0.5 2 2 2 2 1
1 2 ⱖ4 4 2 2 0.5 2 2 2 2 0.25 2 4 2 2 0.5
2 2 2 2 2 2 1 2 2 ⱖ2 2 0.125 1 ⱖ2 1 ⱖ2 1
ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32
ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32
ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32 16 ⱖ32 ⱖ32 ⱖ32 ⱖ32 ⱖ32
1 1 1 1 0.5 1 1 1 2 2 1 1 1 2 1 ⱖ32 2
ⱖ8/152 4/76 0.25/4.75 2/38 1/19 0.25/4.75 1/19 ⱖ8/152 4/76 8/152 1/19 1/19 4/76 8/152 0.25/4.75 ⱖ8/152 1/19
Phenotypeb
PMLTC PMLTC PMLT PMLTC PMLTc PMLT pMLTc PMLTC PMLTC PMLTC PMLTc pMLTc PMLTC PMLTC PMLT PMLTCF pMLTc
a Breakpoints (intermediate and resistant, respectively) according to the Clinical Laboratory Standards Institute: telithromycin (TEL), 2 and ⱖ4 g/ml; penicillin G (PEN), 0.1 to 1 and ⱖ2 g/ml; amoxicillin (AMX), 4 and ⱖ8 g/ml; cefotaxime (CTX 关nonmeningitis兴), 2 and ⱖ4 g/ml; clarithromycin (CLR), 0.5 g/ml and ⱖ1 g/ml; clindamycin (CLI), 0.5 and ⱖ1 g/ml; tetracycline (TET), 4 and ⱖ8 g/ml; levofloxacin (LVX), 4 and ⱖ8 g/ml; trimethoprim-sulfamethoxazole (SXT), 1/19 to 2/38 g/ml and ⱖ4/76 g/ml. All isolates were susceptible to vancomycin (MIC of ⱕ1 g/ml). ND, no data. b Resistance phenotypes: p, penicillin G intermediate; P, penicillin G resistant; M, macrolide resistant; L, lincosamide resistant; T, tetracycline resistant; c, cotrimoxazole intermediate; C, cotrimoxazole resistant; F, fluoroquinolone resistant. c BAL, bronchoalveolar lavage.
⫺5, ⫺6, ⫺7, ⫺28, ⫺33, and ⫺90 [upstream of the ATG start codon of erm(B)]. These mutations were homologous to mutations found at positions 320, 319, 318, 297, and 292 in Tn1545, respectively. In the open reading frame of erm(B), the
mutation E58A was observed in 1 isolate, I75T was observed in 9 isolates, N100S was observed in 13 isolates, H118R was observed in 9 isolates, and V171A was observed in 2 isolates. However, some of the mutations described in the present study
TABLE 2. Genotypic and phenotypic characterization and macrolide resistance mechanisms of 17 telithromycin-nonsusceptible S. pneumoniae strains from Europe Alteration(s) in: Strain
Serotype
ST a
Genotype
Phenotype
23S rRNAb
Ribosomal protein L4
T107G, A1513T, T1514A, A1530T, T1531A, A1535G, A1745T C150T, A1745T, C2216T C150T, A1745T, C2216T C150T, A1745T A138G, A260G, A1745T A138G, A260G, A1745T
WT c
S20N
I75T, H118R
WT WT WT WT WT
S20N S20N S20N WT WT
WT WT WT WT WT
WT WT S20N WT WT
N100S N100S I75T, H118R N100S E58A, I75T, N100S, H118R N100S N100S I75T, H118R N100S I75T, H118R
WT WT
WT S20N
Pn 302
15A
63
erm(B)
cMLSB
PW PW PW PW PW
19A 19A 23F 14 14
81 81 81 143 143
erm(B) erm(B) erm(B) erm(B) erm(B)
cMLSB cMLSB cMLSB cMLSB cMLSB
PW 521 PW 899 PW 1760 PW 1765 PS 5646
14 14 14 14 14
143 143 143 143 143
erm(B) erm(B) erm(B) erm(B) erm(B)
cMLSB cMLSB cMLSB cMLSB cMLSB
PW 1840 PW 1420
14 14
156 276
erm(B) erm(B)
cMLSB cMLSB
A138G, A260G, A1745T A138G, A260G, A1745T C150T A138G, A260G, A1745T T107G; A138G; A260G, C389T, A1745T A138G; A260G, A1745T C150T
PW 1791
19A
276
erm(B)
cMLSB
C150T
WT
S20N
PW 735 PW 778 PW 868
19A 19A 14
1558 1569 2010
erm(B) erm(B) erm(B)
cMLSB cMLSB cMLSB
C150T, A1745T, C2216T A138G, A260G, A1745T A138G, A260G, A1745T
WT WT WT
S20N S20N WT
712 715 1981 1976 1996
Erm(B)
L22
I75T, N100S, H118R I75T, N100S, H118R, V171A I75T, N100S, H118R, V171A N100S I75T, N100S, H118R N100S
a The STs had the following alleles: ST 63, 2-5-36-12-17-21-14; ST81, 4-4-2-4-4-1-1; ST143, 7-5-10-18-6-8-1; ST 1569, 8-11-10-1-6-8-1; ST276, 2-19-2-17-6-22-14; ST 1558, 7-13-8-12-9-1-1; ST 2010, 7-5-140-18-6-8-215. b Mutations were found in all four copies of the 23S rRNA. c WT, wild type.
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TABLE 3. Genetic relatedness of telithromycin-nonsusceptible S. pneumoniae strains from Europe Strain(s) in present study
Sequence type
Country (present study)
Described serotype(s)a
Country or countries in other reportsb
Portugal, Spain, Italy, Sweden Worldwide Poland, Hungary, United States United Kingdom, Germany, France, Portugal The Netherlands
PN 302 PW 712, PW 715, PW 1981 PW 1976, PW 1996, PW 521, PW 899, PW 1760, PW 1765, PS 5646 PW 778
63 81 143
Germany France, Spain France, Germany
15A 23F, 19A, 19F, 14 14
1569c
Belgium
14, 9V, 3, 9A
PW PW PW PW
276 1558c 156 2010c
Switzerland, France Spain France France
19A 19A 14 14
a b c
1420, PW 1791 735 1840 868
Worldwide
Time
1992–1998 Since 1980s 1994–2002 1997–2003 1997 2001 1988–2003 2001
Serotypes observed in the present study are given in boldface. As available in the MLST database (www.mlst.net) by April 2006. First described in the present study.
are likely not involved in resistance and are outside of regions that are known to interact with the ribosome. Therefore, these mutations may simply represent heterogeneity in the erm(B) gene and its promoter region. To our knowledge, the present report describes the largest collection of telithromycin-nonsusceptible clinical isolates characterized for their resistance mechanisms and their epidemiological background to date. The global surveillance project PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) reported that only 0.2% of S. pneumoniae isolates collected between 1999 and 2003 were resistant to telithromycin and that all showed the cMLSB phenotype (10). Concordantly, the present study and other studies on telithromycin resistance in S. pneumoniae report on resistance mainly in erm(B)-positive strains. In addition, a recent study showed that telithromycin can be removed from Streptococcus pyogenes by efflux, related to the presence of the mef gene (3). Faccone and coworkers have reported on a high-level telithromycin-resistant isolate from Argentina. The isolate was mef(E) positive, but erm(B) and erm(A) negative (9). In addition, substantial levels of telithromycin resistance were recently detected in a collection of soil isolates, possibly due to inactivation of the compound (6).
Analysis of the clonal relatedness of the present collection of strains showed that telithromycin resistance was predominantly found among two clones, ST 81 and ST 143. ST 81 (Spain23F-1 clone and its serotype 19A variant) is known to be responsible for the worldwide spread of resistance among pneumococci (19). In summary, the present study demonstrates that mutations in the erm(B) gene, its promoter region, ribosomal protein L4, and 23S rRNA may be associated with resistance to telithromycin in clinical isolates of S. pneumoniae. Although the incidence of telithromycin resistance in pneumococci remains rare worldwide, the spread of telithromycin resistance to multidrug resistance clones with worldwide distribution is a worrisome finding of the present study. Nucleotide sequence accession numbers. The GenBank accession numbers of the ermB (upstream region and open reading frame) sequences of this study are DQ855638 (PW1791 and PW1420), DQ855639 (PW899, PW1765, PW1976, and PW521), DQ855640 (PW1840), DQ855641 (PW712 and PW715), DQ855642 (PW1760), DQ855643 (PW735), DQ855644 (PW788), DQ855645 (PW868), DQ855646 (Pn302), DQ855647 (PW1981), DQ855648 (PS5646), and DQ855649 (PW1996).
FIG. 1. Schematic representation of alterations of the regulatory sequences of erm(B) genes from isolates of S. sanguis (pAM77); S. pneumoniae containing Tn1545; and the clinical isolates PW 1840, PW 1981, PW 712, PW 715, and PW 735. The other 12 isolates show the Tn1545-like organization (27 amino acids). SD1 and SD2 (Shine-Dalgarno sites) represent the ribosomal binding sites for the control peptide and the erm(B) gene. ⫺10 and ⫺35 represent promoter regions. The numbers in the boxes represent the length of the open reading frame of the leader peptide. The nucleotides in boldface given at the end of the leader peptides represent insertions as compared to pAM77. The resulting sequence up to the next stop codon is given.
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The authors thank Sandra Barbosa for excellent technical assistance. We thank the PneumoWorld Study Group for cooperation and for providing the isolates. This study was supported in part by grant RKI-415/1369235 from the German Ministry of Health (Bundesminister fu ¨r Gesundheit) and by the German Ministry for Science and Technology (BMFT) (CAP net). REFERENCES 1. Ackermann, G., and A. C. Rodloff. 2003. Drugs of the 21st century: telithromycin (HMR 3647)—the first ketolide. J. Antimicrob. Chemother. 51:497– 511. 1a.Al-Lahham, A., and R. R. Reinert 2005. Molecular characterization and time-kill kinetics of Streptococcus pneumoniae isolates with reduced susceptibility to telithromycin, abstract C1-1418, p. 82. 45th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, D.C. 2. Appelbaum, P. C. 2002. Resistance among Streptococcus pneumoniae: implications for drug selection. Clin. Infect. Dis. 34:1613–1620. 3. Canton, R., A. Mazzariol, M. I. Morosini, F. Baquero, and G. Cornaglia. 2005. Telithromycin activity is reduced by efflux in Streptococcus pyogenes. J. Antimicrob. Chemother. 55:489–495. 4. 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. 5. Clinical Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing: 15th informational supplement. Clinical Laboratory Standards Institute, Wayne, Pa. 6. D’Costa, V. M., K. M. McGrann, D. W. Hughes, and G. D. Wright. 2006. Sampling the antibiotic resistome. Science 311:374–377. 7. Douthwaite, S., J. Jalava, and L. Jakobsen. 2005. Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm. Mol. Microbiol. 58:613–622. 8. 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. 9. Faccone, D., P. Andres, M. Galas, M. Tokumoto, A. Rosato, and A. Corso. 2005. Emergence of a Streptococcus pneumoniae clinical isolate highly resistant to telithromycin and fluoroquinolones. J. Clin. Microbiol. 43:5800–5803. 10. Farrell, D. J., and D. Felmingham. 2004. Activities of telithromycin against 13,874 Streptococcus pneumoniae isolates collected between 1999 and 2003. Antimicrob. Agents Chemother. 48:1882–1884. 11. Hisanaga, T., D. J. Hoban, and G. G. Zhanel. 2005. Mechanisms of resis-
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