Methyltransferase Erm(37) slips on rRNA to confer atypical resistance in Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.     

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.     

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.     

RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA^II enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA^II, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA^II in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA^II activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA^II, thereby facilitating TEL binding to the ribosome.     

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.     

Synthesis and antibacterial activity of novel C12 ethyl ketolides

A novel series of C(12) ethyl erythromycin derivatives have been discovered which exhibit in vitro and in vivo potency against key respiratory pathogens, including those resistant to erythromycin. The C(12) modification involves replacing the natural C(12) methyl group in the erythromycin core with an ethyl group via chemical synthesis. From the C(12) ethyl macrolide core, a series of C(12) ethyl ketolides were prepared and tested for antibacterial activity against a panel of relevant clinical isolates. Several compounds were found to be potent against macrolide-sensitive and -resistant bacteria, whether resistance was due to ribosome methylation (erm) or efflux (mef). In particular, the C(12) ethyl ketolides 4k,4s,4q,4m, and 4t showed a similar antimicrobial spectrum and comparable activity to the commercial ketolide telithromycin. The in vivo efficacy of several C(12) ethyl ketolides was demonstrated in a mouse infection model with Streptococcus pneumoniae as pathogen.     

<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.     

Conformations in solution and bound to bacterial ribosomes of ketolides, HMR 3647 (telithromycin) and RU 72366: a new class of highly potent antibacterials

The new class of antibiotics called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. Further modifications of the 3 keto-macrolactone backbone led to 11,12-hydrazonocarbamate ketolides with an imidazolyl pyridine chain: the file-leader of ketolide class, HMR 3647 (telithromycin), and its N-bis-demethyl-derivative, RU 72366. The potency of HMR 3647 is higher than that of RU 72366. Stereospecific 1H and 13C resonance assignments of HMR 3647 and RU 72366 have been determined and have allowed a detailed quantitative conformational analysis of the uncomplexed form of the molecules. The comparative conformation of HMR 3647 in solution and its N-bis-demethyl-derivative in D2O has been carried out using different heteronuclear correlation experiments in conjunction with nuclear Overhauser effect experiments and in particular long-range 3J(CH) coupling constants and using molecular dynamics (MD) methods. The study of ketolide ribosome interaction has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The database of ribosome-bound ketolide structures has been used to compare the structure(s) of ketolide in ribosome-ketolide complexes with the conformational preferences of free ketolides and to highlight the significant differences between HMR 3647 and RU 72366. A comparison of the conformations bound to ribosome was made with those of other previously studied ketolide (RU 004) and macrolides and would explain the remarkable potencies of HMR 3647 in inhibiting protein synthesis.     

High rates of macrolide resistance among clinical isolates of Streptococcus agalactiae in Tunisia

One hundred sixty non duplicate erythromycin resistant Streptococcus agalactiae isolates were collected in Tunisia from January 2005 to December 2007 They were investigated to determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. agalactiae isolates were isolated from urinary specimens (38.75%, 62/160). The constitutive MLSB phenotype (cMLS) showed in 84.3% (135/160) with high MICs of macrolides and lincosamides (MIC90>256 microg/mL) and 8.2% (13/160) inducible MLSB phenotype (iMLS) with high MICs of macrolides (MIC90>256 microg/mL) and moderately increased MICs of lincosamides (MIC90=8 microg/mL). The M phenotype showed in 7.5% (12/160) with moderately increased MICs of macrolides (MIC90=32 microg/mL) and low MICs of lincosamides (MIC90=0.75 microg/mL). All strains were susceptible to quinupristun-dalfopristin association and linezolid (MIC90: 05 and 0.38 microg/mL respectively). Strains with MLSB phenotype harboured erm(B) gene with 825% (n=132), erm(TR) gene with 8.12% (n=13) and erm(B) plus mef (A) with 1.88% (n=3). All strains categorized as M phenotype carried the mef(A) gene (75%, n=12). cMLSB phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent.     

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide- resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E. coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA . Furthermore, intracellular accumulation of [¹⁴C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.     

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.     

Structural insight into the antibiotic action of telithromycin against resistant mutants

The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.     

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.     

Novel hybrids of 15-membered 8a- and 9a-azahomoerythromycin A ketolides and quinolones as potent antibacterials

A series of novel 6-O-substituted and 6,12-di-O-substituted 8a-aza-8a-homoerythromycin A and 9a-aza-9a-homoerythromycin A ketolides were synthesized and evaluated for in vitro antibacterial activity against a panel of representative erythromycin-susceptible and erythromycin-resistant test strains. Another series of ketolides based on 14-membered erythromycin oxime scaffold was also synthesized and their antibacterial activity compared to those of 15-membered azahomoerythromycin analogues. In general, structure-activity studies have shown that 14-membered ketolides displayed favorable antibacterial activity in comparison to their corresponding 15-membered analogues within 9a-azahomoerythromycin series. However, within 8a-azahomoerythromycin series, some compounds incorporating a ketolide combined with either quinoline or quinolone pharmacophore substructures showed significantly potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLS(B))-resistant Gram-positive pathogens as well as fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display hitherto unprecedented in vitro activity against the constitutively MLS(B)-resistant strain of Staphylococcus aureus. In addition, they also represent an improvement over telithromycin (2) and cethromycin (3) against fastidious Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis.     

Macrolide resistance and <Emphasis Type="Italic">In Vitro</Emphasis> selection of resistance to antibiotics in <Emphasis Type="Italic">Lactobacillus</Emphasis> isolates

Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.     

Synthesis and antibacterial activity of C11, C12-cyclic urea analogues of ketolides

C11, C12-cyclic urea analogues of ketolides were designed and synthesized by use of a novel ketene acetal intermediate. This intermediate enabled introduction of an amino group at C12 stereospecifically and in high yield. The resulting cyclic urea ketolides appear to have in vitro activity similar to that of telithromycin which contains a C11, C12 cyclic carbamate moiety. Some of the C2 fluorinated compounds have improved potency against erm-containing Streptococcus pyogenes.     

The macrolide-ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA

The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L-cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl-aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70-fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10-fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.     

The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.     

A family of r-determinants in Streptomyces spp. that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics.

Inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics in Streptomyces spp. comprises a family of diverse phenotypes in which characteristic subsets of the macrolide-lincosamide-streptogramin antibiotics induce resistance mediated by mono- or dimethylation of adenine, or both, in 23S ribosomal ribonucleic acid. In these studies, diverse patterns of induction specificity in Streptomyces and associated ribosomal ribonucleic acid changes are described. In Streptomyces fradiae NRRL 2702 erythromycin induced resistance to vernamycin B, whereas in Streptomyces hygroscopicus IFO 12995, the reverse was found: vernamycin B induced resistance to erythromycin. In a Streptomyces viridochromogenes (NRRL 2860) model system studied in detail, tylosin induced resistance to erythromycin associated with N6-monomethylation of 23S ribosomal ribonucleic acid, whereas in Staphylococcus aureus, erythromycin induced resistance to tylosin mediated by N6-dimethylation of adenine. Inducible macrolide-lincosamide-streptogramin resistance was found in S. fradiae NRRL 2702 and S. hygroscopicus IFO 12995, which synthesize the macrolides tylosin and maridomycin, respectively, as well as in the lincosamide producer Streptomyces lincolnensis NRRL 2936 and the streptogramin type B producer Streptomyces diastaticus NRRL 2560. A wide range of different macrolides including chalcomycin, tylosin, and cirramycin induced resistance when tested in an appropriate system. Lincomycin was active as inducer in S. lincolnensis, the organism by which it is produced, and streptogramin type B antibiotics induced resistance in S. fradiae, S. hygroscopicus, and the streptogramin type B producer S. diastaticus. Patterns of adenine methylation found included (i) lincomycin-induced monomethylation in S. lincolnensis (and constitutive monomethylation in a mutant selected with maridomycin), (ii) concurrent equimolar levels of adenine mono- plus dimethylation in S. hygroscopicus, (iii) monomethylation in S. fradiae (and dimethylation in a mutant selected with erythromycin), and (iv) adenine dimethylation in S. diastaticus induced by ostreogrycin B.     

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