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.     

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.     

Characteristic expression of three genes,msr(A), mph(C) and erm(Y), that confer resistance to macrolide antibiotics on Staphylococcus aureus

We have reported that the gene mph(C) (formally referred to as 'mphBM') is located on plasmid pMS97 342 bp downstream of the msr(A) gene. msr(A) specifies resistance to macrolides by ABC-transporter-mediated efflux, and mph(C) has 49% identity to the amino acid sequence of MPH(2')II, which encodes a phosphotransferase that inactivates some macrolide antibiotics. A strain of Staphylococcus aureus NCTC8325 containing plasmid pMS97 inactivated unlabeled and (14)C-labeled erythromycin when tested by bioautographic and radioautographic techniques. In addition to erythromycin, other 14-membered ring macrolides (except for ketolides), 15-membered ring macrolides and 16-membered ring macrolides, mycinamicin, rosamicin and YM133, were inactivated by the strain. Erythromycin inactivation products produced by the strain carrying pMS97 were completely different from those produced by Escherichia coli BM694 bearing plasmid pAT63, which contains the ereA gene encoding an esterase that hydrolyzes macrolide lactones. Constructs formed with the msr(A) and mph(C) genes, and with the msr(A), mph(C) and erm(Y) genes, showed erythromycin-inactivating activity, but another construct built with the mph(C) gene alone failed to show such activity. This result suggests that any region of the msr(A) gene is needed for the expression of mph(C).     

Macrolide Resistance in Staphylococcus aureus

A heat-inducible mutant, resistant to macrolide antibiotics (Mac), was isolated from Staphylococcus aureus MS537 in which Mac-resistance was induced by subinhibitory concentrations of erythromycin (EM). After induction at 42 C, this mutant acquired a high resistance to both Mac and lincomycin (LMC). Transduction and biochemical studies revealed that spiramycin (SP)-resistance in this mutant was induced by exposure to a high temperature (42 C) or by treatment with EM in broth but not in phosphate buffer. Induction did not take place when chloramphenicol (CM) was added to the induction mixture. Ribosomes from the mutant cultured at 42 C decreased their affinity for SP and consequently polypeptide synthesis on such ribosomes was not inhibited by SP, when compared with those cultured at 30 C. From these results, it was concluded that alteration of ribosomes took place after induction by exposure at high temperatures or by EM-treatment and that the mechanism of SP-resistance after induction was accounted for by a decrease in SP-binding to ribosomes.     

Inducible ribosomal RNA methylation in Streptomyces lividans, conferring resistance to lincomycin

Streptomyces lividans TK21 possesses inducible ribosomal RNA methylase activity that confers high-level resistance to lincomycin and lower levels of resistance to certain macrolides. The methylase gene (designated lrm) is inducible by erythromycin and other macrolides and also by celesticetin (a lincosamide) but not by lincomycin. The lrm enzyme monomethylates the N6-amino group of adenosine at position 2058 within 23S-like ribosomal RNA.     

Gene lmrB of Corynebacterium glutamicum confers efflux-mediated resistance to lincomycin

The lmrB gene of Corynebacterium glutamicum, which confers specific resistance to lincosamides, such as lincomycin and clindamycin, was isolated. C. glutamicum cells, carrying the lmrB gene in a multicopy plasmid, showed increased resistance to lincomycin with a MIC of 230 microg/ml, which is a 9-fold increase compared to that of the wild type. The lmrB-disrupted mutant became sensitive to the compound. No difference in sensitivity to erythromycin, penicillin G, tetracycline, chloramphenicol, spectinomycin, nalidixic acid, gentamicin, streptomycin, ethidium bromide, and sodium dodecyl sulfate was observed. The protonophore carbonyl cyanide m-chlorophenylhydrazone abolished the lincomycin-resistance of lmrB-carrying cells. The putative protein product of the gene contained 14-transmembrane regions and showed high amino acid-sequence homology to the drug efflux pumps of other organisms. In addition, the putative protein contained a motif for major facilitators, suggesting a role in efflux-mediated resistance to lincomycin.     

Novel mutants of 23S RNA: characterization of functional properties.

Single point mutations corresponding to the positions G2505 and G2583 have been constructed in the gene encoding E.coli 23S rRNA. These mutations were linked to the second mutation A1067 to T, known to confer resistance to thiostrepton (1). Mutant ribosomes were analyzed in vitro for their ability to direct poly(U) dependent translation, their missence error frequency and in addition their sensitivity to peptidyltransferase inhibitors. It was evident that the mutated ribosomes had an altered dependence on [Mg2+] and an increased sensitivity to chloramphenicol during poly(U) directed poly(Phe) synthesis. In a transpeptidation assay mutated ribosomes were as sensitive to chloramphenicol as wild-type ribosomes. However, the mutant ribosomes exhibited an increased sensitivity to lincomycin. An increase in translational accuracy was attributed to the mutations at the position 2583: accuracy increased in the order G less than A less than U less than C.     

Site of action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics

The enzyme which confers resistance to erythromycin in the producing organism Streptomyces erythraeus dimethylates a single adenine residue in Bacillus stearothermophilus 23 S rRNA. This corresponds to residue Ade 2058 in Escherichia coli 23 S RNA. The methylase responsible for resistance to macrolides, lincomycin, and streptogramin B-related antibiotics in Staphylococcus aureus also acts at this site.     

Drug Resistance of Staphylococci

Examination of cross resistance to macrolide antibiotics in erythromycin resistant staphylococcal strains isolated from clinical sources in the United States showed that there were two types of cross resistance, group A (13.4%) and group C (86.6%). Group A possessed multiple resistance to the macrolide antibiotics, erythromycin, oleandomycin, leucomycin and spiramycin, and was also resistant to lincomycin. In group C, resistance to erythromycin alone or to both erythromycin and oleandomycin could be induced by exposure to erythromycin.     

Erythromycin-inducible resistance in Staphylococcus aureus: requirements for induction

At least two functionally different types of ribosomes are found in strains of Staphylococcus aureus which display "dissociated" resistance to erythromycin. One type of ribosome is found under conditions of growth in ordinary nutrient broth, and the second is formed during growth in the presence of erythromycin. In these strains, erythromycin acts as an inducer of resistance to three different classes of inhibitors of the 50S ribosomal subunit-the macrolides, lincosamides, and streptogramin B-type antibiotics. The optimal inducing concentration of erythromycin is between 10(-8) and 10(-7)m. Concentrations as low as 10(-9)m can produce a 10-fold increase in resistant cells over the uninduced, background level, whereas concentrations greater than 10(-7)m block induction owing to inhibition of protein synthesis. Resistant cells begin to appear within 5 to 10 min after addition of erythromycin (to 10(-7)m), and within 40 min (i.e., about one generation) more than 90% of the entire culture is resistant to erythromycin as well as to lincomycin and vernamycin B(alpha). A resistant culture becomes sensitive if grown for 90 min in the absence of erythromycin. The process of induction is inhibited by chloramphenicol and streptovaricin, which inhibit protein and ribonucleic acid synthesis, respectively, but not by novobiocin, which inhibits deoxyribonucleic acid synthesis. Resistant cells produced in this manner fail to concentrate (14)C-erythromycin and (14)C-lincomycin, but not (14)C-chloramphenicol. Constitutively erythromycin-resistant strains which do not require the presence of erythromycin for expression of resistance can be selected on media containing antibiotics which belong to any one of the three classes. Two patterns of constitutive resistance have been found. These are (i) generalized constitutive resistance-which involves resistance in the absence of erythromycin to all members of each of the three cited classes of 50S subunit inhibitors which were tested, and (ii) partial constitutive resistance-which involves different degrees of resistance, in the absence of erythromycin, to various members of the three classes. Several different patterns of variable constitutivity are possible. 50S ribosomal subunits isolated from induced or constitutively resistant cells show decreased ability to bind erythromycin and lincomycin, and possible enzymatic inactivation of these antibiotics has been rigorously excluded. The induced change, therefore involves modification of ribosome structure rather than modification of the antibiotic.     

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.     

Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome

Summary Several mutations conferring resistance to streptomycin, kanamycin, spectinomycin, erythromycin, and lincomycin on the group A streptococcal strain 56188 have been mapped by two- and three-point crosses using transduction with bacteriophage A25. The markers are located in two linkage regions too distant to be cotransduced. One harbors the streptomycin and kanamycin loci which are transduced jointly at 78% and the other bears loci for spectinomycin (spc), erythromycin (eryA), and lincomycin (linA) resistance, in this order. spc and linA are cotransduced at a frequency of about 27%. Analysis of three-point crosses involving spc-4, eryA300, and linA12 according to the Wu model for random general transduction shows consistency of the theoretical predictions with the experimental data and indicates that the intervals of the above sequence are about 22% and 6% of the average length of the transduced piece.     

Methicillin-resistant Staphylococcus aureus (MRSA) in the German Democratic Republic. Incidence and strain-characteristics

In GDR methicillin-resistance strains of S. Aureus only occur in connection with nosocomial infections with a comparably low incidence (about 2%). They are not found in outpatients. For the detection of MRSA the test on nutrient medium L4 with addition of 5% NaCl has proved successful. All of the MRSA exhibit a rather unique pattern of strain-characteristics; they are nontypable by the basic-set-phages and show a reaction with the experimental phage A 994. The MRSA are multiple drug-resistant (generally penicillins, cephalosporins, isoxyzolylpenicillins, oxytetracycline, minocycline, streptomycin, erythromycin, lincomycin and additionally chloramphenicol and gentamycin, kanamycin, tobramycin). The genetical characterization and the plasmid-pattern analysis has shown that only resistance to chloramphenicol and in one case also to macrolides are determined by plasmids (MW 2.0 and 1.8 Megadalton). The determinants for the other resistance-characters are obviously located on the chromosome. Altogether these data indicate that the MRSA described are derivatives of a single-strain-clone.     

Sensitivity of the causative agent of tularemia to antibiotics

Sensitivity of the tularemia causative agent of different geographical races to antibiotics such as streptomycin, tetracycline, gentamicin, rifampicin (20 strains), ampicillin, polymyxin M, erythromycin, oleandomycin (361 strains) and lincomycin (294 strains) was studied. High sensitivity of the tularemi a microbe to streptomycin, tetracycline, rifampicin (MIC of 10 gamma/ml), gentamicin (MIC of 1 gamma/ml) and resistance to 50 gamma of ampicillin and 1000 gamma/ml of polymyxin M were found. Combined use of 50 gamma of ampicillin and 100 gamma/ml of polymyxin M added to the nutrient medium for growth inhibition of the foreign flora on isolation of the tularemia causative agent from the infected material including stable laboratory animal carcases was recommended. Marked differences in sensitivity of the strains of different geographical races to the macrolides and lincomycin were observed. The strains of the non-Arctic and Central Asiatic races were of low resistance to the above drugs (the MIC of erythromycin, oleandomycin and lincomycin were 10--50, 50--400 and 25--100 gamma/ml respectively. Within the holarctic race 40 per cent were low resistant and 60 per cent were highly resistant to these drugs. The above drugs should not be used for treatment of tularemia cases.     

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.     

The Bifidobacterium longum NCIMB 702259T ctr gene codes for a novel cholate transporter

Preexposure of Bifidobacterium longum NCIMB 702259T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [14C]cholate.     

Sensitivity of Corynebacterium diphtheriae isolated in Saint-Petersburg to antibacterial drugs]

One hundred and thirty strains of Corynebacterium diphtheriae isolated in St. Petersburg (42 toxigenic and 88 nontoxigenic) were tested with the method of serial dilutions in solid media for their susceptibility to 20 antibacterial drugs. The MICs of the drugs for the isolates ranged from < or = 0.015 to > or = 32.0 micrograms/ml. 13 per cent of the isolates was resistant at least to one antibacterial drug. The isolates resistant to erythromycin (11.5 per cent), lincomycin (11.5 per cent) and trimethoprim (8.5 per cent) were most frequent. 3 isolates (2.3 per cent) had multiple resistance to 8 drugs: benzylpenicillin, ampicillin, oxacillin, chloramphenicol, erythromycin, lincomycin, trimethoprim, and nitroxolin. No significant differences in the susceptibility of the toxigenic and nontoxigenic strains of C. diphtheriae were detected. Gentamicin, rifampicin, tetracycline, doxycycline and pefloxacin were the most active antibiotics.     

Carbomycin resistance in mouse L cells

A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.