Macrolide and Ketolide Antibiotics Inhibit the Cytotoxic Effect of Trastuzumab Emtansine in HER2-Positive Breast Cancer Cells: Implication of a Potential Drug-ADC Interaction in Cancer Chemotherapy.

Macrolides are widely used for the long-term treatment of infections and chronic inflammatory diseases. The pharmacokinetic features of macrolides include extensive tissue distribution because of favorable membrane permeability and accumulation within lysosomes. Trastuzumab emtansine (T-DM1), a HER2-targeting antibody-drug conjugate (ADC), is catabolized in the lysosomes, where Lys-SMCC-DM1, a potent cytotoxic agent, is processed by proteinase degradation and subsequently released from the lysosomes to the cytoplasm through the lysosomal membrane transporter SLC46A3, resulting in an antitumor effect. We recently demonstrated that erythromycin and clarithromycin inhibit SLC46A3 and attenuate the cytotoxicity of T-DM1; however, the effect of other macrolides and ketolides has not been determined. In this study, we evaluated the effect of macrolide and ketolide antibiotics on T-DM1 cytotoxicity in a human breast cancer cell line, KPL-4. Macrolides used in the clinic, such as roxithromycin, azithromycin, and josamycin, as well as solithromycin, a ketolide under clinical development, significantly attenuated T-DM1 cytotoxicity in addition to erythromycin and clarithromycin. Of these, azithromycin was the most potent inhibitor of T-DM1 efficacy. These antibiotics significantly inhibited the transport function of SLC46A3 in a concentration-dependent manner. Moreover, these compounds extensively accumulated in the lysosomes at the levels estimated to be 0.41-13.6 mM when cells were incubated with them at a 2 µM concentration. The immunofluorescence staining of trastuzumab revealed that azithromycin and solithromycin inhibit the degradation of T-DM1 in the lysosomes. These results suggest that the attenuation of T-DM1 cytotoxicity by macrolide and ketolide antibiotics involves their lysosomal accumulation and results in their greater lysosomal concentrations to inhibit the SLC46A3 function and T-DM1 degradation. This suggests a potential drug-ADC interaction during cancer chemotherapy.

Simultaneous determination of novel ketolide antibiotic nafithromycin and its major metabolite in human plasma using liquid chromatography tandem mass spectrometry.

Aim: A sensitive method to quantify nafithromycin and its N-desmethyl metabolite in human plasma was necessary for Phase I pharmacokinetic studies. Methodology: A precise and accurate LC-MS/MS bioanalytical method has been developed and validated for the simultaneous quantification of nafithromycin (NFT, WCK 4873) and N-desmethyl metabolite (M1, WCK 4978) in human plasma. Clarithromycin was used as an internal standard. Protein precipitation technique was used as sample preparation approach. The calibration curve was linear (r ≥ 0.99) over the concentration range of 10-5000 ng/ml for NFT and M1. Method was validated as per US FDA guideline. Conclusion: The proposed method was successfully applied for determination of plasma levels of the NFT and M1 during Phase I clinical studies.

Erythromycin, Cethromycin and Solithromycin display similar binding affinities to the E. coli's ribosome: A molecular simulation study.

Macrolide antibiotics bind to the exit tunnel of the ribosome and inhibit protein synthesis blocking its translocation. Thus, antibiotics including the known macrolide Erythromycin (ERY) are active against bacteria. However, at present, some bacteria show resistance to drugs, which requires the development of new powerful antibacterial agents. One possible way is to use the ERY structure, but change its side chains, while the size of the lactone ring can remain unchanged or change. In this work we consider Cethromycin (CET) and Solithromycin (SOL), which are ketolides with quinolylallyl group at C6 and aminophenyl at C11, respectively (both of them have the same lactone ring as ERY). Experiments have shown that these ketolides have improved efficacy against pathogens, but their binding affinity to the E. coli's ribosome is almost identical. To clarify this issue, we have studied in detail the binding mechanisms of ERY, CET and SOL using the docking and molecular dynamic simulations. In agreement with the experiments, we showed that these compounds have similar binding affinities. Desosamine and lactone ring groups play a critical role in the binding of ERY to the ribosome. In CET and SOL, the contribution of keto and alkylaryl groups is balanced by cyclic carbamate. We have demonstrated that increased fluctuations in the ribosomal residues at the binding site led to an increase in the entropic term in the free binding energy of ERY compared to SOL and CET. The alkyl-aryl arm of both ketolides strongly interacts with A752 and U2609. In addition, the presence of macrolides in the exit tunnel can alter the conformation of U2585, which is located in the peptidyl transferase center, through non-bonded interaction. Therefore, the side chain of ketolides affects not only the binding site but also other residues possibly leading to a strong effect on the protein synthesis process. We predict that to combat bacterial mutations, it is necessary either to design a bulk and charged group as a cladinose, or to use several groups with different signs of charges. This prediction can be used for the development of new efficient antibiotics.

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations.

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1'-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes.

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action.

Differences between the structures of bacterial, archaeal, and eukaryotic ribosomes account for the selective action of antibiotics. Even minor variations in the structure of ribosomes of different bacterial species may lead to idiosyncratic, species-specific interactions of the drugs with their targets. Although crystallographic structures of antibiotics bound to the peptidyl transferase center or the exit tunnel of archaeal (Haloarcula marismortui) and bacterial (Deinococcus radiodurans) large ribosomal subunits have been reported, it remains unclear whether the interactions of antibiotics with these ribosomes accurately reflect those with the ribosomes of pathogenic bacteria. Here we report X-ray crystal structures of the Escherichia coli ribosome in complexes with clinically important antibiotics of four major classes, including the macrolide erythromycin, the ketolide telithromycin, the lincosamide clindamycin, and a phenicol, chloramphenicol, at resolutions of ∼3.3 Å-3.4 Å. Binding modes of three of these antibiotics show important variations compared to the previously determined structures. Biochemical and structural evidence also indicates that interactions of telithromycin with the E. coli ribosome more closely resembles drug binding to ribosomes of bacterial pathogens. The present data further argue that the identity of nucleotides 752, 2609, and 2055 of 23S ribosomal RNA explain in part the spectrum and selectivity of antibiotic action.

Revisiting the structures of several antibiotics bound to the bacterial ribosome.

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

Different effects of telithromycin on MUC5AC production induced by human neutrophil peptide-1 or lipopolysaccharide in NCI-H292 cells compared with azithromycin and clarithromycin.

OBJECTIVES: Mucus hypersecretion is a prominent feature in patients with chronic respiratory tract infections such as cystic fibrosis and diffuse panbronchiolitis, and the clinical effectiveness of macrolide antibiotics has been reported in these patients. Because human neutrophil peptide-1 (HNP-1), an antimicrobial peptide in neutrophils, exists in high concentrations in the airway fluid of these patients, we examined the direct effect of HNP-1 on MUC5AC mucin production using NCI-H292 cells. The effects of macrolide antibiotics on the response were also examined. METHODS: MUC5AC synthesis was assayed using RT-PCR and ELISA. Phosphorylation of ERK1/2 was determined by western blotting. RESULTS: Stimulation with HNP-1 or lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa increases the production of MUC5AC mRNA and protein, and an additive effect was found upon co-stimulation with both HNP-1 and LPS. Azithromycin and clarithromycin had inhibitory effects on overproduction of MUC5AC induced by HNP-1 or LPS stimulation. Telithromycin also had an inhibitory effect on MUC5AC production induced by LPS, but not on production by HNP-1. Phosphorylation of ERK1/2 was induced by HNP-1 or LPS stimulation, and azithromycin, clarithromycin and telithromycin had inhibitory effects on ERK1/2 phosphorylation induced by LPS, but not by HNP-1. CONCLUSIONS: These findings suggest that neutrophil-derived defensins as bacterial components contribute to excessive mucus production in patients with respiratory tract infections, and that macrolide and ketolide antibiotics directly inhibit these actions by interfering with intracellular signal transduction. However, the mechanism of telithromycin inhibition of MUC5AC synthesis may differ from the response induced by azithromycin and clarithromycin.

The intracellular accumulation of phagocytic and epithelial cells and the inhibitory effect on Chlamydophila (Chlamydia) pneumoniae of telithromycin and comparator antimicrobials.

The intracellular accumulation of telithromycin was measured and compared with ciprofloxacin, clarithromycin, minocycline and erythromycin. The activities of telithromycin, clarithromycin and minocycline against Chlamydophila (Chlamydia) pneumoniae were compared in an intracellular killing assay. Maximal telithromycin accumulation (mean intracellular/extracellular concentration ratio) ranged from 6.7 (A549 lung epithelial cells) to 11.8 (THP-1 monocytic cells). This ratio was similar to that of clarithromycin, but less than for minocycline. Minimum inhibitory and minimum lethal telithromycin concentrations against six clinical strains of C. pneumoniae were <0.015-0.03 mg/L and 0.03-0.06 mg/L, respectively, which were lower than those found for minocycline. These results show that telithromycin accumulates rapidly into epithelial cells, similar to previously reported results for phagocytic cells. Intracellular accumulation of telithromycin was lower than that observed for minocycline, but telithromycin demonstrated substantially more potent antichlamydial activity.

Benefit-risk assessment of telithromycin in the treatment of community-acquired pneumonia.

The purpose of this review is to assess the benefits and risks associated with the use of the ketolide antibacterial telithromycin, currently licensed for the treatment of adults with mild to moderate community-acquired pneumonia (CAP). Telithromycin is active against both the major (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis) and atypical/intracellular (Chlamydophila pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae) CAP pathogens. It is associated with a low potential to select for resistance and has maintained its in vitro activity against isolates of respiratory pathogens in countries where it has been in clinical use for several years. In randomized clinical trials, telithromycin has demonstrated efficacy comparable to the established antibacterial classes (macrolides, fluoroquinolones and beta-lactams) in the treatment of CAP.The safety profile of telithromycin is broadly similar to that of other antibacterials used to treat CAP. The most common adverse events are gastrointestinal adverse effects and headache; these are generally mild to moderate in severity and reversible. Telithromycin appears to be well tolerated by adult patients in all age groups, including those with co-morbid conditions. In common with other antibacterials, telithromycin has the potential to affect the corrected QT interval; the concomitant use of cisapride or pimozide with telithromycin is contraindicated, while telithromycin should be avoided in patients receiving Class IA or Class III antiarrhythmic drugs. Visual disturbances (usually transient) have occurred in a small proportion of patients treated with telithromycin; it is recommended that activities such as driving are minimized during treatment. Telithromycin is contraindicated in patients with myasthenia gravis. Hepatic dysfunction may occur in some patients taking telithromycin; rare cases of acute hepatic failure and severe liver injury, including deaths, have been reported. As telithromycin is an inhibitor of the cytochrome P450 (CYP) 3A4 system, coadministration of telithromycin with drugs metabolized by this pathway may require dose adjustments (e.g. with benzodiazepines) or a temporary hiatus in the use of the coadministered drug (e.g. HMG-CoA reductase inhibitors) metabolized by CYP3A4. Telithromycin may potentiate the effects of oral anticoagulants; careful monitoring is recommended in patients receiving telithromycin and oral anticoagulants simultaneously.Although serious and sometimes fatal events have occurred in patients receiving telithromycin therapy, current data indicate that telithromycin offers an acceptable benefit risk ratio in the treatment of mild to moderate CAP.

Ketolides: pharmacological profile and rational positioning in the treatment of respiratory tract infections.

Ketolides differ from macrolides by removal of the 3-O-cladinose (replaced by a keto group), a 11,12- or 6,11-cyclic moiety and a heteroaryl-alkyl side chain attached to the macrocyclic ring through a suitable linker. These modifications allow for anchoring at two distinct binding sites in the 23S rRNA (increasing activity against erythromycin-susceptible strains and maintaining activity towards Streptococcus pneumoniae resistant to erythromycin A by ribosomal methylation), and make ketolides less prone to induce methylase expression and less susceptible to efflux in S. pneumoniae. Combined with an advantageous pharmacokinetic profile (good oral bioavailability and penetration in the respiratory tract tissues and fluids; prolonged half-life allowing for once-a-day administration), these antimicrobial properties make ketolides an attractive alternative for the treatment of severe respiratory tract infections such as pneumonia in areas with significant resistance to conventional macrolides. For telithromycin (the only registered ketolide so far), pharmacodynamic considerations suggest optimal efficacy for isolates with minimum inhibitory concentration values < or = 0.25 mg/l (pharmacodynamic/pharmacokinetic breakpoint), calling for continuous and careful surveys of bacterial susceptibility. Postmarketing surveillance studies have evidenced rare, but severe, side effects (hepatotoxicity, respiratory failure in patients with myasthenia gravis, visual disturbance and QTc prolongation in combination with other drugs). On these bases, telithromycin indications have been recently restricted by the US FDA to community-acquired pneumonia, and caution in patients at risk has been advocated by the European authorities. Should these side effects be class related, they may hinder the development of other ketolides such as cethromycin (in Phase III, but on hold in the US) or EDP-420 (Phase II).

Increasing telithromycin resistance among Streptococcus pyogenes in Europe.

OBJECTIVES: To assess changes in macrolide and ketolide resistance among Streptococcus pyogenes in Europe and to examine the relationship of resistance to antimicrobial usage. METHODS: Clinical S. pyogenes isolates were collected from Denmark, Finland, France, Germany, Italy, Netherlands, Norway, Spain, Sweden, UK, Croatia, Hungary, Poland, Slovak Republic and Slovenia during 2002-03 (n = 2165) and 2004-05 (n = 2333). Resistance to telithromycin (MIC > or = 2) and erythromycin (MIC > or = 0.5) was determined by CLSI broth microdilution. Changes in resistance over time and the relationship of resistance to antimicrobial use (European Surveillance of Antimicrobial Consumption data) were assessed. Telithromycin-resistant isolates were characterized by PFGE to determine genetic relatedness and by PCR to detect mef(A), erm(A) and erm(B). RESULTS: The erythromycin resistance rate during 2004-05 (11.6%) was similar to 2002-03 (10.4%). The proportion of macrolide-resistant isolates with the constitutive MLS(B) phenotype increased from 29.3% (2002-03) to 45.7% (2004-05). Telithromycin resistance increased from 1.8% in 2002-03 to 5.2% in 2004-05. For Western Europe, associations of telithromycin and erythromycin resistance, respectively, were found with azithromycin use (R2 = 0.52 and 0.60), clarithromycin use (R2 = 0.76 and 0.85) and total macrolide/lincosamide use (R2 = 0.75 and 0.69). For Eastern Europe, associations of antimicrobial use with resistance were not apparent. The 162 telithromycin-resistant isolates comprised 42 PFGE patterns with 68.5% in eight major PFGE groups. The erm(B) gene was detected in 155 of the 162 telithromycin-resistant isolates. CONCLUSIONS: Significant increases in telithromycin resistance occurred from 2002-03 to 2004-05 in Europe. Macrolide use appears to be a factor in the emergence of ketolide resistance among S. pyogenes in Western Europe.

Effects of acute renal failure on the pharmacokinetics of telithromycin in rats: negligible effects of increase in CYP3A1 on the metabolism of telithromycin.

It was reported that the expression of CYP3A1 increased in rats with acute renal failure induced by uranyl nitrate (rat model of U-ARF) compared with controls. It was shown that telithromycin was mainly metabolized via CYP3A1/2 in rats in this study. Hence, the pharmacokinetic parameters of telithromycin were compared after both intravenous and oral administration at a dose of 50 mg/kg to control rats and a rat model of U-ARF. After intravenous administration of telithromycin to rats with U-ARF, the AUC and renal clearance (Cl(r)) were significantly greater (35.0% increase) and slower (99.1% decrease), respectively, than the controls. Unexpectedly, the nonrenal clearance (Cl(nr)) of telithromycin was comparable between the two groups of rats, suggesting that CYP3A isozyme responsible for the metabolism of telithromycin seemed not to be expressed considerably in the rat model of U-ARF. After oral administration of telithromycin to rats with U-ARF, the AUC was also significantly greater (127% increase) than the controls and the value, 127%, was considerably greater than 35.0% after intravenous administration of telithromycin. This may be due mainly to the decrease in the intestinal first-pass effect of telithromycin compared with controls in addition to significantly slower Cl(r) than controls.

Resistance phenotypes conferred by macrolide phosphotransferases.

This study aims to compare the resistance phenotypes conferred by various genes encoding enzymes that phosphorylate erythromycin. The mph genes were cloned into Escherichia coli AG100A susceptible to macrolides and ketolides following disruption of the AcrAB pump. An 882 bp sequence containing a premature stop codon, homologous to the three other previously described mph genes and present widely among Enterobacteriaceae, was found to confer resistance to erythromycin by phosphorylation. The mph(C) gene, as reported for mph(B), also conferred resistance to spiramycin. The mph(A) gene was unique in conferring resistance to azithromycin. The four investigated genes conferred resistance to telithromycin.

The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3.

Macrolides may cause severe drug interactions due to the inhibition of metabolizing enzymes. Transporter-mediated uptake of drugs into cells [e.g., by members of the human organic anion transporting polypeptide (OATP) family] is a determinant of drug disposition and a prerequisite for subsequent metabolism. However whether macrolides are also inhibitors of uptake transporters, thereby providing an additional mechanism of drug interactions, has not been systematically studied. The human OATP family members OATP1B1 and OATP1B3 mediate the uptake of endogenous substances and drugs such as antibiotics and HMG-CoA reductase inhibitors (statins) into hepatocytes. In this study we investigated the potential role of these uptake transporters on macrolide-induced drug interactions. By using sulfobromophthalein (BSP) and the HMG-CoA reductase inhibitor pravastatin as substrates, the effects of the macrolides azithromycin, clarithromycin, erythromycin, and roxithromycin and of the ketolide telithromycin on the OATP1B1- and OATP1B3-mediated uptake were analyzed. These experiments demonstrated that the OATP1B1- and OATP1B3-mediated uptake of BSP and pravastatin can be inhibited by increasing concentrations of all macrolides except azithromycin. The IC50 values for the inhibition of OATP1B3-mediated BSP uptake were 11 microM for telithromycin, 32 microM for clarithromycin, 34 microM for erythromycin, and 37 microM for roxithromycin. These IC50 values were lower than the IC50 values for inhibition of OATP1B1-mediated BSP uptake (96-217 microM). These macrolides also inhibited in a concentration-dependent manner the OATP1B1- and OATP1B3-mediated uptake of pravastatin. In summary, these results indicate that alterations of uptake transporter function by certain macrolides/ketolides have to be considered as a potential additional mechanism underlying drug-drug interactions.

Sampling the antibiotic resistome.

Microbial resistance to antibiotics currently spans all known classes of natural and synthetic compounds. It has not only hindered our treatment of infections but also dramatically reshaped drug discovery, yet its origins have not been systematically studied. Soil-dwelling bacteria produce and encounter a myriad of antibiotics, evolving corresponding sensing and evading strategies. They are a reservoir of resistance determinants that can be mobilized into the microbial community. Study of this reservoir could provide an early warning system for future clinically relevant antibiotic resistance mechanisms.

Species-specific antibiotic-ribosome interactions: implications for drug development.

In the cell, the protein synthetic machinery is a highly complex apparatus that offers many potential sites for functional interference and therefore represents a major target for antibiotics. The recent plethora of crystal structures of ribosomal subunits in complex with various antibiotics has provided unparalleled insight into their mode of interaction and inhibition. However, differences in the conformation, orientation and position of some of these drugs bound to ribosomal subunits of Deinococcus radiodurans (D50S) compared to Haloarcula marismortui (H50S) have raised questions regarding the species specificity of binding. Revisiting the structural data for the bacterial D50S-antibiotic complexes reveals that the mode of binding of the macrolides, ketolides, streptogramins and lincosamides is generally similar to that observed in the archaeal H50S structures. However, small discrepancies are observed, predominantly resulting from species-specific differences in the ribosomal proteins and rRNA constituting the drug-binding sites. Understanding how these small alterations at the binding site influence interaction with the drug will be essential for rational design of more potent inhibitors.

Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm.

Macrolide and ketolide antibiotics inhibit protein synthesis on the bacterial ribosome. Resistance to these antibiotics is conferred by dimethylation at 23S rRNA nucleotide A2058 within the ribosomal binding site. This form of resistance is encoded by erm dimethyltransferase genes, and is found in many pathogenic bacteria. Clinical isolates of Streptococcus pneumoniae with constitutive erm(B) and Streptococcus pyogenes with constitutive erm(A) subtype (TR) are resistant to macrolides, but remain susceptible to ketolides such as telithromycin. Paradoxically, some strains of S. pyogenes that possess an identical erm(B) gene are clinically resistant to ketolides as well as macrolides. Here we explore the molecular basis for the differences in these streptococcal strains using mass spectrometry to determine the methylation status of their rRNAs. We find a correlation between the levels of A2058-dimethylation and ketolide resistance, and dimethylation is greatest in S. pyogenes strains expressing erm(B). In constitutive erm strains that are ketolide-sensitive, appreciable proportions of the rRNA remain monomethylated. Incubation of these strains with subinhibitory amounts of the macrolide erythromycin increases the proportion of dimethylated A2058 (in a manner comparable with inducible erm strains) and reduces ketolide susceptibility. The designation 'constitutive' should thus be applied with some reservation for most streptococcal erm strains. One strain worthy of the constitutive designation is S. pyogenes isolate KuoR21, which has lost part of the regulatory region upstream of erm(B). In S. pyogenes KuoR21, nucleotide A2058 is fully dimethylated under all growth conditions, and this strain displays the highest resistance to telithromycin (MIC > 64 microg ml-1).