Mechanisms of resistance to telithromycin in Streptococcus pneumoniae

Tamiko Hisanaga1,*, Daryl J. Hoban1,2 and George G. Zhanel1,2,3

1 Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; 2 Department of Clinical Microbiology, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba, R3A 1R9, Canada; 3 Department of Medicine, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba, R3A 1R9, Canada


* Corresponding author. Tel: +1-204-787-4684; Fax: +1-204-787-4699; E-mail: thisanaga{at}shaw.ca


    Abstract
 Top
 Abstract
 References
 
Reports of ketolide resistance remain scarce, however, a few laboratory-derived and clinical isolates of resistant Streptococcus pneumoniae have been documented. Mutations in key telithromycin-binding sites such as domains II and V of the 23S rRNA and ribosomal proteins L4 and L22, as well as mutations of the resistance determinant erm(B) are associated with elevated telithromycin MICs. Mutations in the secondary binding site of domain II coupled with ribosomal methylation may have serious resistance consequences should the domain II binding site be lost. Although ketolides are purported to maintain excellent activity against efflux-positive isolates, laboratory-derived telithromycin-resistant strains have been generated. As telithromycin usage increases, ketolide-resistant isolates of S. pneumoniae may well increase.

Keywords: mutations , 23S rRNA , mef(A) , erm(B)

Streptococcus pneumoniae is an important human pathogen that has been identified as a primary cause of community-acquired pneumonia (CAP).1 Macrolides are commonly used to treat CAP, however, macrolide resistance is increasing worldwide. The two major mechanisms by which S. pneumoniae become resistant to macrolides are target modification and macrolide efflux. The erm(B) gene encodes a methyl transferase that dimethylates A2058 of the 23S rRNA, a key binding residue, conferring high-level (MIC ≥ 32 mg/L) macrolide resistance.2 Efflux pumps encoded by either of two genetic determinants, Tn1207.1 and the macrolide efflux genetic assembly (MEGA), confer resistance to 14- and 15-membered macrolides.3 Tn1207.1 is a defective conjugative transposon encoding the mef(A) variant of the efflux gene, whereas MEGA encodes the mef(E) gene, with the two genes sharing 90% identity.

Ketolides are macrolide derivatives designed to overcome macrolide resistance. Like macrolides, ketolides bind to the bacterial ribosome near the peptide exit tunnel which is composed largely of domains II and V, blocking the tunnel and inhibiting peptide elongation.2,4 The peptide exit tunnel is composed largely of RNA, but at its narrowest point a 12 Å constriction is formed by the ribosomal proteins L4 and L22.5 Unlike macrolides, ketolides do not act as inducers of macrolide, lincosamide, streptogramin B (MLSB) resistance and do not appear to be affected by efflux.6 The global surveillance project PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) reported that 99.8% of S. pneumoniae isolates from 1999 to 2003 were susceptible to telithromycin.7 Thus, although ketolide resistance is rare, resistant isolates have nonetheless been documented.

Using the NCCLS provisional breakpoints for telithromycin resistance of ≥4 mg/L the majority of laboratory-derived mutants exhibited frank resistance to telithromycin, although a number required elevated MICs that indicated reduced susceptibility to telithromycin rather than resistance (Table 1).8 One mutant with a single base deletion (A752) in a stretch of highly conserved adenines in hairpin loop 35 of domain II, was resistant to telithromycin, showing a 500-fold increase in MIC.8 This mutant is notable in that it possesses the mutation in domain II at a novel binding site for ketolides, which, with their alkyl-aryl side chains, are able to bind to an additional site along domain II, providing improved activity and greater binding affinity than existing macrolides.2 Although A752 does not form a direct contact with the drug, this base is protected in chemical footprinting studies, indicating its importance in binding the drug.9 The deletion may have resulted in structural changes to the domain II ribosomal binding site by disrupting helix 35.2,10 This is consistent with a report that a single mutation (U754A) in E. coli was sufficient to render the isolate resistant to telithromycin. Further, an E. coli mutation in domain V (U2609C) resulted in resistance solely to ketolides and not macrolides.9 This base is located behind A752 of domain II and mutation of this base may generate resistance to ketolides by bringing about a conformational change around A752 such that the ketolide may no longer bind to domain II.


View this table:
[in this window]
[in a new window]
 
Table 1. S. pneumoniae laboratory-derived mutants conferring decreased susceptibility or resistance to telithromycin

 
A notable laboratory-derived mutant contained a deletion of the upstream region of erm(B) resulting in a highly resistant strain.11 Previous deletions in the erm(B) attenuator region have been associated with change from inducible to constitutive resistance in streptococci.12 The deletions removed inverted repeats whose secondary structure normally sequesters the ribosome binding site. Clinical isolates with mutations in the erm(B) leader sequences have also been noted that display either decreased susceptibility or resistance to telithromycin (Table 2). Clinical isolates with a 136 bp deletion in the erm(B) promoter region which removed the second Shine-Dalgarno (SD2) site produced a new fusion protein with the remainder of the control peptide and the erm(B) gene under the control of SD1.13 A few isolates contained additional mutations that may have further served to decrease their susceptibility to telithromycin. The latest PROTEKT report identified only 10 telithromycin-resistant isolates, all of which possessed the erm(B) methylase gene.7 However, these isolates were not examined for mutations and it is uncertain whether the erm(B) methylase gene alone could be responsible for resistance.


View this table:
[in this window]
[in a new window]
 
Table 2. S. pneumoniae clinical isolates exhibiting decreased susceptibility or resistance to telithromycin

 
Although telithromycin-resistant pneumococci with mutations in the erm(B) gene or its promoter appear to exhibit higher MICs than pneumococci bearing other mutations, clinical isolates with mutations in the ribosomal proteins have also been reported. An L4 ribosomal insertion of six amino acids when transformed into a ketolide-susceptible isolate was accompanied by a 500-fold increase in MIC.14 The isolate had a 60% increase in generation time which probably rendered it unstable as a transformant bearing this mutation reverted to wild-type, both in respect to MICs and removal of the mutation as assessed by DNA sequencing, after 80 generations in the absence of antibiotic. In one instance, pneumococcal macrolide failure selected for telithromycin-resistant isolates with an insertion in L22, one of which additionally contained an A2058 mutation.15

Efflux-positive S. pneumoniae isolates that were rendered telithromycin-resistant (MICs 2–8 mg/L) were found to contain no mutations in the 23S rRNA regions, or in mef(A), L4 and L22 genes.11 When the antibiotic pressure was removed, the MICs decreased by two- to eightfold, with only one isolate remaining telithromycin-resistant. This is similar to findings reported by Davies et al.16 in which mutants derived from mef(E) parent strains returned to their original MICs (or close to that) after 10 passages on antibiotic free-media. Upon subsequent challenge with 1 or 2 mg/L of telithromycin, a mutant reverted to having an MIC of 8 mg/L, indicating selective pressure with telithromycin is required if S. pneumoniae with the mef(A) gene are to maintain their telithromycin MICs.11

Using radiolabelled telithromycin, efflux of the drug was clearly demonstrated in Streptococcus pyogenes isolates expressing the mef(A) gene, resulting in reduced telithromycin activity.17 S. pyogenes isolates containing the mef(A) were generally telithromycin-susceptible (MIC range 0.06–4; mode 1 mg/L). While ketolide efflux has not been demonstrated in S. pneumoniae, the ketolide examined in such experiments was cethromycin, with CCCP (carbonyl cyanide M-chlorophenylhydrazone) used as an efflux inhibitor. In contrast, the previous experiment differed in that inhibition of efflux was studied using telithromycin as the substrate with sodium arsenate as the inhibitor.6,17 Furthermore, the efflux resistance determinant Tn1207.1 of S. pneumoniae is part of a larger conjugative transposon, Tn1207.3 that carries mef(A) in clinical isolates of S. pyogenes.18 The mechanism of action for these mutants showing efflux probably involves overexpression of the pump in the presence of antibiotic. It is interesting to note that in general the telithromycin MIC values for efflux-positive isolates are higher than the MIC values for erm(B)-positive isolates.2

Telithromycin resistance can be induced by macrolides, but generally only to low levels. A notable exception was reported by Canu et al.8 who derived mutants by serial passage of five isolates of S. pneumoniae to four macrolides and telithromycin. The only mutant showing telithromycin resistance displayed an A752 deletion and had been selected for by exposure to clarithromycin. Telithromycin exposure did not result in any telithromycin-resistant isolates. The previously mentioned macrolide treatment failure was associated with a mutant with high-level telithromycin resistance (8–16 mg/L) following intravenous and oral clarithromycin treatment.15

Generally, the more mutations an organism has, the more resistant it becomes. The pneumococcal isolate exhibiting the highest telithromycin resistance reported to date (256 mg/L) had mutations in the erm(B) gene, the erm(B) control region and the ribosomal protein L4.19 A recent study by Novotny et al.20 investigated the impact of dual mutations at key 23S rRNA telithromycin binding sites; A752 of domain II and A2058 of domain V in E. coli. Base substitutions at A752 and an A752 deletion or insertion resulted in low-level telithromycin resistance, with MICs of 15–20, 25 and 20 mg/L, respectively. Base substitution of adenine at position 2058 with a guanine results in the addition of bulky substituents similar to methylation of A2058 by erm(B), resulting in high-level telithromycin resistance (200 mg/L).10,20 When mutations at 2058 were combined with domain II mutations, resistance to telithromycin no longer occurred. These results led the authors to conclude that mutations within domain II whether alone or in combination with an A2058 mutation confer significant resistance to telithromycin. The high level of telithromycin resistance for this A2058G mutant, however, is not in agreement with clinical and laboratory-derived mutants of S. pneumoniae. Seven clinical isolates with all four rrn operons possessing an A2058G mutation were still susceptible to telithromycin, with MICs ranging from 0.12 to 0.5 mg/L.13

Although the incidence of telithromycin resistance remains rare, a number of laboratory-derived and clinical S. pneumoniae isolates have been reported that exhibit elevated telithromycin MICs. Mutations in the erm(B) gene and its promoter region, ribosomal proteins L4 and L22 and in domains II and V of the 23S rRNA have resulted in decreased susceptibility, or in some cases resistance to telithromycin and are likely to represent a harbinger of things to come. Mutations in domain II should be of concern as alterations could potentially result in loss of this binding site. When coupled with ribosomal methylation, high levels of ketolide resistance in S. pneumoniae could prevail. Of particular interest are the potential of mef-positive telithromycin-resistant S. pneumoniae and the increasing numbers of clinical isolates with mutations in the erm(B) control region. The relatively low numbers of telithromycin-resistant isolates have resulted in a lack of understanding regarding some of the underlying mechanisms of telithromycin resistance. The need for further studies of telithromycin-resistant pneumococci and their mechanisms of resistance is warranted.


    References
 Top
 Abstract
 References
 
1. Jedrzejas MJ. Pneumococcal virulence factors: structure and function. Microbiol Mol Biol Rev 2001; 65: 187–207.[Abstract/Free Full Text]

2. Zhanel GG, Hisanaga T, Nichol K et al. Ketolides: an emerging treatment for macrolide-resistant respiratory infections, focusing on Streptococcus pneumoniae. Expert Opin Emerg Drugs 2003; 8: 297–321.[CrossRef][Medline]

3. Edelstein PH. Pneumococcal resistance to macrolides, lincosamides, ketolides and streptogramin B agents: molecular mechanisms and resistance phenotypes. Clin Infect Dis 2004; 38: S322–7.[CrossRef][ISI][Medline]

4. Schlunzen F, Harms JM, Franceschi F et al. Structural basis for the antibiotic activity of ketolides and azalides. Structure (Cambridge) 2003; 11: 329–38.

5. Nissen P, Hansen J, Ban N et al. The structural basis of ribosome activity in peptide bond synthesis. Science 2000; 289: 920–30.[Abstract/Free Full Text]

6. Capobianco JO, Cao Z, Shortridge VD et al. Studies of the novel ketolide ABT-773: transport, binding to ribosomes, and inhibition of protein synthesis in Streptococcus pneumoniae. Antimicrob Agents Chemother 2000; 44: 1562–7.[Abstract/Free Full Text]

7. Farrell DJ, Felmingham D. Activities of telithromycin against 13,874 Streptococcus pneumoniae isolates collected between 1999 and 2003. Antimicrob Agents Chemother 2004; 48: 1882–4.[Abstract/Free Full Text]

8. Canu A, Malbruny B, Coquemont M et al. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: 125–31.[Abstract/Free Full Text]

9. Garza-Ramos G, Xiong L, Zhong P et al. Binding site of macrolide antibiotics on the ribosome: new resistance mutation identifies a specific interaction of ketolides with rRNA. J Bacteriol 2001; 183: 6898–907.[Abstract/Free Full Text]

10. Franceschi F, Kanyo Z, Sherer EC et al. Macrolide resistance from the ribosome perspective. Curr Drug Targets Infect Disord 2004; 4: 177–91.[CrossRef][Medline]

11. Walsh F, Willcock J, Amyes S. High-level telithromycin resistance in laboratory-generated mutants of Streptococcus pneumoniae. J Antimicrob Chemother 2003; 52: 345–53.[Abstract/Free Full Text]

12. Rosato A, Vicarini H, Leclercq R. Inducible or constitutive expression of resistance in clinical isolates of streptococci and enterococci cross-resistant to erythromycin and lincomycin. J Antimicrob Chemother 1999; 43: 559–62.[Abstract/Free Full Text]

13. Farrell DJ, Morrissey I, Bakker S et al. In vitro activities of telithromycin, linezolid, and quinupristin–dalfopristin against Streptococcus pneumoniae with macrolide resistance due to ribosomal mutations. Antimicrob Agents Chemother 2004; 48: 3169–71.[Abstract/Free Full Text]

14. Tait-Kamradt A, Davies T, Appelbaum PC et al. Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob Agents Chemother 2000; 44: 3395–401.[Abstract/Free Full Text]

15. Perez-Trallero E, Marimon JM, Iglesias L et al. Fluoroquinolone and macrolide treatment failure in pneumococcal pneumonia and selection of multidrug-resistant isolates. Emerg Infect Dis 2003; 9: 1159–62.[ISI][Medline]

16. Davies T, Dewasse B, Jacobs MR et al. In vitro development of resistance to telithromycin (HMR 3647), four macrolides, clindamycin and pristinamycin in Streptococcus pneumoniae. Antimicrob Agents Chemother 2000; 44: 414–7.[Abstract/Free Full Text]

17. Cornaglia G, Mazzriol A, Zuliani J et al. Telithromycin activity is reduced by efflux in Streptococcus pyogenes. In: Programs and Abstracts of the 12th European Congress of Clinical Microbiology and Infectious Diseases, Milan, Italy, 2002. Abstract P473. European Society of Clinical Microbiology and Infectious Diseases.

18. Santagati M, Iannelli F, Cascone C et al. The novel conjugative transposon tn1207.3 carries the macrolide efflux gene mef(A) in Streptococcus pyogenes. Microb Drug Resist 2003; 9: 243–7.[CrossRef][ISI][Medline]

19. Tait-Kamradt A, Reinert RR, Al-Lahham A et al. High-level ketolide-resistant streptococci. In: Programs and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract C1-1813, p. 101, American Society for Microbiology, Washington, DC, USA.

20. Novotny GW, Jakobsen L, Andersen NM et al. Ketolide antimicrobial activity persists after disruption of interactions with domain II of 23S rRNA. Antimicrob Agents Chemother 2004; 48: 3677–83.[Abstract/Free Full Text]

21. Farrell DJ, Morrissey I, Bakker S et al. Mutations in erm(B) associated with rare, low-level telithromycin resistance in Streptococcus pneumoniae: 3-year data from PROTEKT. In: Programs and Abstracts of the 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic, 2004. Abstract P1465. European Society of Clinical Microbiology and Infectious Diseases.

22. Pihlajamaki M, Jalava J, Huovinen P et al. Antimicrobial resistance of invasive pneumococci in Finland in 1999–2000. Antimicrob Agents Chemother 2003; 47: 1832–5.[Abstract/Free Full Text]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
56/3/447    most recent
dki249v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Hisanaga, T.
Articles by Zhanel, G. G.
PubMed
PubMed Citation
Articles by Hisanaga, T.
Articles by Zhanel, G. G.