Macrolide Resistance Conferred by Base Substitutions in 23S rRNA

Resistance to all major groups of antibiotics has arisen hand in hand with their extensive use in medicine and animal husbandry, and macrolide antibiotics are no exception. The therapeutic utility of macrolides has been severely compromised by the emergence of drug resistance in many pathogenic bacteria. The molecular mechanisms by which bacteria become resistant are manifold, but in general these can be collectively characterized as involving either drug efflux, drug inactivation, or alterations in the drug target site. The target site for macrolides is the large (50S) subunit of the bacterial ribosome. Many cases of macrolide resistance in clinical strains can be linked to alteration of specific nucleotides in 23S rRNA within the large ribosomal subunit. Macrolides are natural polyketide products of secondary metabolism in many actinomycete species (51, 140). Clinically useful macrolides consist of a 14-, 15-, or 16-member lactone ring (Table ​(Table1)1) that is generally substituted with two or more neutral and/or amino sugars (16). The structures of the 14- and 16-member-ring macrolides erythromycin and tylosin and of some semisynthetic erythromycin derivatives are shown in Fig. ​Fig.1. 1. The inhibitory action of erythromycin, and probably that of the other 14-member-ring macrolides, is effected at the early stages of protein synthesis when the drug blocks the growth of the nascent peptide chain (7, 140), presumably causing premature dissociation of the peptidyl-tRNA from the ribosome (85). The antimicrobial action of these drugs is compounded by their inhibition of the assembly of new large ribosomal subunits, which leads to gradual depletion of functional ribosomes in the cell (23). The mode of action of the 16-member-ring macrolides is less well characterized, although it is clear that they bind to the same region of the large subunit as the 14-member-ring macrolides and inhibit peptide bond formation in a more direct manner (reviewed in reference 140). TABLE 1 Macrolide antibiotics and their derivatives discussed in this review FIG. 1 Selected clinically important macrolide antibiotics and their derivatives. Two naturally occurring macrolides are shown: erythromycin A, which was the first therapeutic macrolide and possesses a 14-member ring, and tylosin, a 16-member-ring macrolide . Shortly after the introduction of erythromycin in therapy in the 1950s, resistance to the drug was observed in bacterial pathogens (reviewed in reference 76). More disquieting was the observation that erythromycin-resistant strains were cross-resistant not only to all other macrolides but also to the chemically unrelated lincosamide and streptogramin B drugs. This phenomenon was first observed in Staphylococcus aureus and came to be termed the macrolide-lincosamide-streptogramin B (MLSB) antibiotic resistance phenotype. In these S. aureus strains, MLSB resistance can be induced by exposure to low concentrations of erythromycin (151), which leads to expression of a methyltransferase enzyme (ErmC). ErmC specifically methylates 23S rRNA (74) at the N-6 position of adenosine 2058 (A2058) (Escherichia coli numbering) (121), which is a pivotal nucleotide for the binding of MLSB antibiotics (see below). Subsequently, several dozen erm methyltransferase genes have been identified. Many of these are constitutively expressed, and their products all presumably methylate A2058. A new nomenclature system has recently been proposed for the different erm genes, which clarifies their phylogenetic relatedness (105). For a comprehensive account of the action of Erm methyltransferases, see the review by Weisblum (149). Since the discovery of erm genes, another means of resistance involving alteration of rRNA structure has been identified. Under laboratory conditions, single base substitutions introduced into rRNA were shown to confer macrolide resistance. This form of resistance was first observed in the single rRNA (rrn) operon of yeast mitochondria, which was mutated at position A2058 in the large-subunit rRNA (123). Shortly afterwards, similar phenotypes were obtained in E. coli by expression of mutant rrn alleles from multiple-copy plasmids (see, e.g., references 120 and 143). About 6 years ago, reports of rRNA mutations conferring macrolide resistance in clinical pathogens began to appear in the literature. While it is conceptually gratifying to establish that the mutations appearing in pathogens are identical to those previously isolated in laboratory strains, the clinical implications of this are quite disturbing. The 23S rRNA mutations reported so far to cause macrolide resistance are shown in Table ​Table2.2. Generally, pathogenic species that develop macrolide resistance through mutations at A2058 (or neighboring nucleotides) possess only one or two rrn operons, such as in the case of Helicobacter pylori and Mycobacterium species. Resistance in bacteria with multiple rrn operons, such as Enterococcus, Streptococcus, and Staphylococcus species, is generally conferred by Erm methylation of A2058 (Table ​(Table3)3) or by efflux (see e.g., references 70 and 110). However, there are cases of macrolide resistance by drug inactivation (reviewed in reference 150), and there are recent reports of macrolide resistance in Streptococcus pneumoniae strains conferred by mutations in ribosomal proteins L4 and L22 and in rRNA (129; P. Appelbaum, personal communication). Macrolide and ketolide resistance is additionally conferred in E. coli by the expression of small, specific peptides (134), although the level of resistance is probably too low to be a problem in the treatment of clinical strains. TABLE 2 23S rRNA mutations reported to confer macrolide resistance TABLE 3 Macrolide resistance mechanisms found in some pathogens and their numbers of rRNA operons In the following sections of this review, we first look at the current state of knowledge of the bacterial ribosome target site for macrolide antibiotics. A detailed model of a drug target site is a prerequisite for understanding the molecular mechanisms of drug binding and drug resistance and for rational design of new drugs. Our present state of knowledge, although far from being complete, supports the view that the macrolide target site is highly conserved within the ribosomes of all bacteria. We then direct attention to the pathogens, and in particular to H. pylori, that have been shown to attain resistance by rRNA mutation, and we consider the possibility of this form of resistance emerging in other pathogens. Finally, some suggestions are made regarding how future macrolide derivatives might be best equipped to combat bacteria with resistant rRNAs.