Current Drug Targets - Infectious Disorders - Volume 4, Issue 3, 2004

The clonality and mechanisms of macrolide resistance were studied among 345 macrolide resistant Streptococcus pneumoniae strains isolated from 24 countries. The mechanisms of macrolide resistance, serotypes and PFGE types of the strains were determined and representative strains of clones from each country were typed by MLST. Among strains tested 215 had the erm(B) gene, 92 the mef(A) gene, 14 had both erm(B) and mef(A), and 24 had alterations in ribosomal proteins [2 with A2059G substitutions in 23S rRNA, 21 with 69GTG71 to TPS change in L4, and one with erm(B) and deletion of leucine at position 68 in L22]. Serogroups 19, 6, and 23, and serotype 14 were the most common serotypes / serogroups. Dissemination of variants of sequence type (ST) 315 and ST156 were observed in Eastern and Central European countries. In Asiatic countries the most common sequence types were variants of ST236 among strains with mef(A) and ST180 among strains with erm(B). Strains with both erm(B) and mef(A) from Mexico and Singapore were variants of ST236. The widespread clone from Slovakia with ribosomal protein L4 mutation was a variant of ST226. Common clones were observed between Europe, Asia, and America. Overall, while serotypes / serogroups of macrolide resistant isolates were limited, multiple PFGE and MLST types were found, with clustering of common clones within countries.

Macrolides are important antibiotics used in treatment of respiratory tract infections in humans. Although some of these compounds have been in use for 50 years, it has not been until the last few years that their mechanism of action and the nature of ribosomal-based resistance could be more fully understood. With the advent of robust crystals of ribosomal 50S subunits, and structural resolution of macrolides and ketolides complexed to either Haloarcula marismortui or Deinococcus radiodurans 50S, the ability to dissect the binding modes and understand resistance at the level of the ribosome became possible. This review article compares the binding features of 14-, 15-, and 16-membered macrolides to that of ketolides telithromycin and ABT-773 as revealed at the atomistic level. Attempts to understand how modifications to 23S rRNA and / or mutations in ribosomal proteins L4 and L22 that have been found to confer resistance in Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae are told from the perspective of the ribosome.

Macrolide, lincosamide and streptogramin B (MLS B) antibiotics are extensively used for the treatment of wide variety of clinically important Gram-positive bacteria. MLS B antibiotics inhibit protein biosynthesis by targeting the peptidyl transferase centre within the 50S ribosomal subunit. The most widespread mechanism of bacterial resistance to MLS B antibiotics, reported early after their introduction into clinical practice is the modification of the target site exhibited by a family of rRNA methyltransferases designated Erm. Using S-adenosyl-L-methionine, Erm enzymes catalyze mono- or dimethylation of a specific adenine residue in the 23S rRNA. The methyl group sterically hinders the MLS B binding site and disrupts the hydrogen bonding between the macrolides and the rRNA, thus rendering bacteria resistant. This review summarizes the current understanding of Erm-mediated resistance, in light of high-resolution structural data of bacterial ribosome and with specific focus on the results of recent genetic, biochemical and structural studies of Erm methyltransferases and their cognate rRNA substrate. Although many features of MLS B resistance remain indistinct, the present knowledge can now serve as the guidance for development of both new antimicrobial drugs and potential inhibitors of Erm enzymes, hence providing a new lead to solve the urgent problem of the macrolide resistance based on the ribosome methylation.

Type M resistance to macrolides in Streptococcus pyogenes and Streptococcus pneumoniae is associated to the presence of macrolide efflux genes (mef). These genes are carried by 3 very conserved chromosomal genetic elements: (i) “mega”, 5.5 kb, typical of S. pneumoniae, carries mef(E), can insert at different sites in the bacterial chromosome and in other genetic elements, does not have genes encoding putative recombinases or transposases, and it is not conjugative; (ii) Tn1207.3, 54, 52 kb, typical of S. pyogenes, carries mef(A), it integrates at a single specific site in the bacterial chromosome, carries 3 ORFs encoding for putative recombinases, it is transferred by conjugation among different streptococcal species, and its genome resembles that of a bacteriophage; (iii) Tn1207.1, 7.2 kb, is a defective form of Tn1207.3 found in a clonal population of S. pneumoniae, it carries mef(A), it integrates at a single specific site in the bacterial chromosome (the same of Tn1207.3), and it is not conjugative.

A number of different mechanisms of macrolide resistance have been described in Gram-negative bacteria. These include 16 acquired genes (esterases, phosphorylases, rRNA methylases, and effluxes) and include those thought to be unique to Gram-negative bacteria (both esterases and two of the phosphorylases) and those shared with Gram-positive bacteria (one phosphorylase) and those primarily of Gram-positive origin (rRNA methylases and efflux genes). In addition, mutations, which modify the 23S rRNA, ribosomal proteins L4 and / or L22, and / or changes in expression of innate efflux systems which occur by missense, deletion and / or insertion events have been described in five Gramnegative groups, while an innate transferase conferring resistance to streptogramin A has been identified in a sixth genus. However, the amount of information on both acquisition and mutations leading to macrolide, lincosamides, streptogramins, ketolides and oxazolidinones (MLSKO) resistance is limited. As a consequence this review likely underestimates the true distribution of acquired genes and mutations in Gram-negative bacteria. As use of these drugs increases, it is likely that interaction between members of the MLSKO antibiotic family and Gram-negative bacteria will continue to change resistance to these antibiotics; by mutations of existing genes as well as by acquisition and perhaps mutations of acquired resistant genes in these organisms and more work needs to be done to get a clearer picture of what is in the Gram-negative population now, such that changes can be monitored.

Inactivation, one of the mechanisms of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics, appears to be fairly rare in clinical isolates in comparison with target site modification or efflux. However, inactivation is one of the major mechanisms through which macrolide-producing organisms avoid self-damage during antibiotic biosynthesis. The inactivation mechanisms for MLS antibiotics in pathogens are mainly hydrolysis, phosphorylation, glycosylation, reduction, deacylation, nucleotidylation, and acetylation. The ere (erythromycin resistance esterase) and mph (macrolide phosphotransferase) genes were originally found in Escherichia coli. Subsequently, Wondrack et al. (Wondrack, L.; Massa, M.; Yang, B.V.; Sutcliffe, J. Antimicrob. Agents Chemother., 1996, 40, 992) reported ere-like activity in Staphylococcus aureus. In addition, a variant of erythromycin esterase was found in Pseudomonas sp. from aquaculture sediment by Kim et al. (Kim, Y.H.; Cha, C.J.; Cerniglia, C.E. FEMS Microbiol. Lett., 2002, 210, 239). Although the mph genes, including mph(K), were first characterized in E. coli, a recent study revealed that S. aureus and Stenotrophomonas maltophilia have mph(C). The mph(C) has a low G+C content, like mph(B), and has high homology with mph(B), but not with mph(A) or mph(K). Consequently, the mph(C) and ere(B) genes seem to have originated from Gram-positive bacteria and been transferred between Gram-positive and Gram-negative bacteria. In this chapter, the genes and the mechanisms involved in the inactivation of MLS antibiotics by antibiotic-producing bacteria are reviewed.

Erythromycin, the first antibacterial macrolide introduced into the clinical setting over 50 years ago, was used extensively not only for the treatment of respiratory tract infections in both adults and children, but also for bone and soft tissue infections, and specific sexually transmitted diseases. Macrolide antibiotics have undergone a dramatic chemical evolution over the past 50 years, culminating in the improved 14- and 16-membered macrolides, acylides and new ketolides. In all cases, improvements in antibacterial activity involved changes in the interplay between the chemical structure of the macrolide and the components of the bacterial cell that dictate ultimate antibacterial activity and efficacy. Target site modification by methylation of ribosomal RNA, the so-called Macrolide-Streptogramin-Lincosamide, (MLS0 resistance and active efflux are the two most common forms of resistance present in the clinic today; however, other resistance mechanisms are known. The first macrolide that bound to MLS-resistant ribosomes was reported in 1989, demonstrating that appropriate structural changes could regain access to the modified ribosome-binding site. In addition, macrolide analogs with reduced affinity for the active efflux pump were identified in 1990, demonstrating that features of pump recognition could be separated from ribosome binding site recognition. Progressive medicinal chemistry led to the synthesis and development of the more recent ketolide class, which combines attributes of both prototypes into one molecule, i.e. non-recognition by the efflux pump and regaining some access to the modified ribosome binding site. Ketolide also lack of induction of erm methylase as do 16-member macrolides. This review will focus on the biological properties of macrolides in terms of their complex interaction with bacterial cells and 50S ribosome target.