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.     

Telithromycin Inhibition of Protein Synthesis and 50S Ribosomal Subunit Formation in <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Cells

The new ketolide antibiotic telithromycin (HMR3647) has been examined for inhibitory effects in cells of Streptococcus pneumoniae. The antibiotic caused a proportional decline in cell growth rate and viability with an IC₅₀ of 15 ng/ml. At a concentration of 7.5 ng/ml, protein synthesis in these cells was reduced by 50%. As seen in other organisms, this compound was also a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays defined the reduced rate of 50S synthesis in antibiotic treated cells. At 7.5 ng/ml the rate was reduced to 50% of the control synthesis rate. An IC₅₀ of 15 ng/ml was found for the effect on this process. 30S ribosomal subunit formation was unaffected by the antibiotic. Inhibition of translation and 50S particle formation are equivalent targets for this antibiotic. The effects of telithromycin in S. pneumoniae are compared with those found in Staphylococcus aureus cells. Received: 29 October 2001 / Accepted: 1 February 2002     

The mechanism of action of macrolides,lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome

The macrolide-lincosamide-streptogramin B class (MLS) of antibiotics contains structurally different but functionally similar drugs, that all bind to the 50S ribosomal subunit. It has been suggested that these compounds block the path by which nascent peptides exit the ribosome. We have studied the mechanisms of action of four macrolides (erythromycin, josamycin, spiramycin and telithromycin), one lincosamide (clindamycin) and one streptogramin B (pristinamycin IA). All these MLS drugs cause dissociation of peptidyl-tRNA from the ribosome. Josamycin, spiramycin and clindamycin, that extend to the peptidyl transferase center, cause dissociation of peptidyl-tRNAs containing two, three or four amino acid residues. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl-tRNAs containing six, seven or eight amino acid residues. Pristinamycin IA causes dissociation of peptidyl-tRNAs with six amino acid residues and telithromycin allows polymerisation of nine or ten amino acid residues before peptidyl-tRNA dissociates. Our data, in combination with previous structural information, suggest a common mode of action for all MLS antibiotics, which is modulated by the space available between the peptidyl transferase center and the drug.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA

Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.     

Binding site of macrolide antibiotics on the ribosome: new resistance mutation identifies a specific interaction of ketolides with rRNA.

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.     

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).     

The ketolide antibiotic ABT-773 is a specific inhibitor of translation and 50S ribosomal subunit formation in Streptococcus pneumoniae cells

ABT-773 is a new 3-keto macrolide antibiotic that has been shown to be very effective against infections by Gram-positive microorganisms. This work examines its inhibitory effects in cells of Streptococcus pneumoniae. ABT-773 caused a proportional decline in cell growth rates and viability with an IC₅₀ of 5 ng/ml. Protein synthesis in these cells was reduced by 50% at an antibiotic concentration of 2.5 ng/ml. This compound was also found to be a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays revealed a reduced rate of 50S synthesis in antibiotic-treated cells. At 2 ng/ml, the rate was reduced to 33% of the control synthesis rate. An IC₅₀ of 5 ng/ml was found for the effect on this process, indicating an equal effect of the drug on translation and assembly. Synthesis of the 30S ribosomal subunit was unaffected by this antibiotic. The effects of ABT-773 in S. pneumoniae are compared with those of the related ketolide antibiotic telithromycin in S. pneumoniae and in Staphylococcus aureus. Received: 6 November 2001 / Accepted: 14 December 2001     

Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance

Crystal structures of H. marismortui large ribosomal subunits containing the mutation G2099A (A2058 in E. coli) with erythromycin, azithromycin, clindamycin, virginiamycin S, and telithromycin bound explain why eubacterial ribosomes containing the mutation A2058G are resistant to them. Azithromycin binds almost identically to both G2099A and wild-type subunits, but the erythromycin affinity increases by more than 10(4)-fold, implying that desolvation of the N2 of G2099 accounts for the low wild-type affinity for macrolides. All macrolides bind similarly to the H. marismortui subunit, but their binding differs significantly from what has been reported in the D. radioidurans subunit. The synergy in the binding of streptogramins A and B appears to result from a reorientation of the base of A2103 (A2062, E. coli) that stacks between them. The structure of large subunit containing a three residue deletion mutant of L22 shows a change in the L22 structure and exit tunnel shape that illuminates its macrolide resistance phenotype.     

Activity of novel inhibitors of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.     

Preferential inhibition of protein synthesis by ketolide antibiotics in Haemophilus influenzae cells

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC(50) values of 1.5 microgram/ml. Protein synthesis was inhibited in cells with a similar IC(50) concentration (1.25 microgram/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism.     

An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

<Emphasis Type="Italic">In Vitro</Emphasis> activity of telithromycin and quinupristin/dalfopristin against methicillin-resistant coagulase-negative staphylococci with defined resistance genotypes

We determined the activities of new antibiotics telithromycin (ketolide) and quinupristin/dalfopristin (streptogramins) against 88 macrolide and/or lincosamide resistant coagulase-negative staphylococci (CoNS) isolates with defined resistance gene status. Telithromycin susceptibility was determined only in erythromycin-sensitive isolates (15) indicating the same mechanisms of resistance. In contrast, all erythromycin-resistant isolates (73) were either constitutively resistant to telithromycin (13 isolates with constitutive erm genes) or demonstrated telithromycin D-shaped zone (60 isolates with inducible msr(A) and/or erm). However, the level of inducible resistance conferred by msr(A) (35 isolates) was borderline even after induction by erythromycin. No quinupristin/dalfopristin resistant isolate was observed if tested by disk-diffusion method (DDM) but 18 isolates were intermediate (MIC = 1-3 mg/L) and two isolates resistant (MIC = 8 mg/L) if tested by E-test. All these isolates were resistant to streptogramin A and harbored vga(A) gene (1 isolate) or vga(A)LC gene (19 isolates). MICs for quinupristin/dalfopristin were higher for isolates with combination of streptogramin A resistance and constitutive MLSB resistance (MIC = 3-8 mg/L in 4 isolates) than for streptogramin A-resistant isolates susceptible to streptogramin B (MIC = 0.5-2 mg/L in 16 isolates). In addition to S. haemolyticus, vga(A)LC was newly identified in S. epidermidis and S. warnerii indicating its widespread occurrence in CoNS. Misidentification of low-level resistant isolates by DDM may contribute to dissemination of streptogramin A resistance.     

Clarithromycin and telithromycin increases interleukin-10 expression in the rat endometriosis model

Endometriosis is a common gynecological disorder associated with infertility. However, treatment options remain limited at present. Since the pathogenesis involves immune responses, the immunomodulatory effect of macrolide on endometriosis has been the focus of much research. A previous study showed that clarithromycin decreased stromal proliferation and promoted apoptosis of fibroblasts in an endometriosis model in rats; however, the mechanism of the effect remains unknown. The aim of this study is to investigate the effect of clarithromycin, one of the major macrolides, and telithromycin, one of the antibiotics belonging to a macrolide group (ketolide), on IL6, IL10 and Ccl2 expression in a rat endometriosis model induced by the surgical transplantation of endometrium onto the peritoneum in 8-week-old female Sprague-Dawley rats. After autotransplantation, the rats were given daily administration of clarithromycin (16 mg/kg/day or telithromycin (12 mg/kg/day) for 3 days. The induced lesions were examined 4 days after autotransplantation. After treatment, IL10 expression in the lesions was increased in rats treated with clarithromycin (1.70-fold) and telithromycin (2.88-fold). The drugs attenuated proliferative stromal lesion of the endometriosis model. The results showed that in the endometriosis model, the drugs enhanced expression of IL10, which may play a role in inhibiting excess inflammatory reaction with its therapeutic effect on the lesion. Macrolide and ketolide therapy may have significant value for the treatment of human endometriosis.     

Incidence, risk factors and mortality of nosocomial pneumonia in Intensive Care Units: A prospective study

Background

Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.

Methods

In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).

Results and discussion

In total, 58.0%, 66.1%, and 55.8% of S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC₅₀ = 0.06 mg/L, MIC₉₀ = 0.12 mg/L).

Conclusion

The high in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.     

Synthesis and antibacterial activity of C-6 carbamate ketolides, a novel series of orally active ketolide antibiotics

A new series of antibacterial ketolides is reported, which features the use of a C-6 carbamate for tethering the arylalkyl sidechain to the macrolide core. The best members of this series display in vitro and in vivo activity comparable to telithromycin. Partial epimerization at C-2, unobserved in previously reported ketolides, was noted for this series.     

Preferential Inhibition of Protein Synthesis by Ketolide Antibiotics in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> Cells

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC₅₀ values of 1.5 μg/ml. Protein synthesis was inhibited in cells with a similar IC₅₀ concentration (1.25 μg/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism. Received: 21 February 2002 / Accepted: 30 April 2002     

Inhibitory action of telithromycin against Shiga toxin and endotoxin

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.