HMR-3004

Antibiotics can cause dormancy (bacteriostasis) or induce death (cidality) of the targeted bacteria. The bactericidal capacity is one of the most important properties of antibacterial agents. However, the understanding of the fundamental differences in the mode of action of bacteriostatic or bactericidal antibiotics, especially those belonging to the same chemical class, is very rudimentary. Here, by examining the activity and binding properties of chemically distinct macrolide inhibitors of translation, we have identified a key difference in their interaction with the ribosome, which correlates with their ability to cause cell death. While bacteriostatic and bactericidal macrolides bind in the nascent peptide exit tunnel of the large ribosomal subunit with comparable affinities, the bactericidal antibiotics dissociate from the ribosome with significantly slower rates. The sluggish dissociation of bactericidal macrolides correlates with the presence in their structure of an extended alkyl-aryl side chain, which establishes idiosyncratic interactions with the ribosomal RNA. Mutations or chemical alterations of the rRNA nucleotides in the drug binding site can protect cells from macrolide-induced killing, even with inhibitor concentrations that significantly exceed those required for cell growth arrest. We propose that the increased translation downtime due to slow dissociation of the antibiotic may damage cells beyond the point where growth can be reinitiated upon the removal of the drug due to depletion of critical components of the gene-expression pathway.

Ketolides, which represent the newest macrolide antibiotics, are generally perceived to be noninducers of inducible erm genes. In the study described in this paper we investigated the effects of several macrolide and ketolide compounds on the expression of the inducible erm (C) gene by Escherichia coli cells. Exposure to 14-member-ring macrolide drugs and to azithromycin led to a rapid and pronounced increase in the extent of dimethylation of Erm(C) target residue A2058 in 23S rRNA. When cells were incubated with subinhibitory concentrations of ketolides, the extent of A2058 dimethylation was also increased, albeit to a lower level and with kinetics slower than those observed with macrolides. The induction of erm (C) expression by ketolides was further confirmed by using a reporter construct which allows the colorimetric detection of induction in a disc diffusion assay. Most of the ketolides tested, including the clinically relevant compounds telithromycin and cethromycin, were able to induce the reporter expression, even though the induction occurred within a more narrow range of concentrations compared to the concentration range at which induction was achieved with the inducing macrolide antibiotics. No induction of the reporter expression was observed with 16-member-ring macrolide antibiotics or with a control drug, chloramphenicol. The deletion of three codons of the erm (C) leader peptide eliminated macrolide-dependent induction but left ketolide-dependent induction unchanged. We conclude that ketolides are generally capable of inducing erm genes. The narrow range of ketolide inducing concentrations, coupled with the slow rate of induction and the lower steady-state level of ribosome methylation, may mask this effect in MIC assays.