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
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Abstract |
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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.
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Efflux-positive S. pneumoniae isolates that were rendered telithromycin-resistant (MICs 28 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 0.064; 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 (816 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 1520, 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.
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