The molecular mechanism of peptide-mediated erythromycin resistance.

The macrolide antibiotic erythromycin binds at the entrance of the nascent peptide exit tunnel of the large ribosomal subunit and blocks synthesis of peptides longer than between six and eight amino acids. Expression of a short open reading frame in 23 S rRNA encoding five amino acids confers resistance to erythromycin by a mechanism that depends strongly on both the sequence and the length of the peptide. In this work we have used a cell-free system for protein synthesis with components of high purity to clarify the molecular basis of the mechanism. We have found that the nascent resistance peptide interacts with erythromycin and destabilizes its interaction with 23 S rRNA. It is, however, in the termination step when the pentapeptide is removed from the peptidyl-tRNA by a class 1 release factor that erythromycin is ejected from the ribosome with high probability. Synthesis of a hexa- or heptapeptide with the same five N-terminal amino acids neither leads to ejection of erythromycin nor to drug resistance. We propose a structural model for the resistance mechanism, which is supported by docking studies. The rate constants obtained from our biochemical experiments are also used to predict the degree of erythromycin resistance conferred by varying levels of resistance peptide expression in living Escherichia coli cells subjected to varying concentrations of erythromycin. These model predictions are compared with experimental observations from growing bacterial cultures, and excellent agreement is found between theoretical prediction and experimental observation.

Adhesive binding of rokitamycin to Staphylococcus aureus ribosomes.

Rokitamycin (RKM), a 3"-O-propionyl derivative of leucomycin A5, is bactericidal against staphylococci near the minimum inhibitory concentrations. RKM bound to ribosomes before-hand is only slightly displaced by erythromycin or josamycin, or even by RKM itself. The adhesive binding of the RKM-ribosome complex might prove to be the lethal event for susceptible staphylococci.

Synergistic bactericidal interaction of josamycin with human neutrophils in vitro.

Josamycin and erythromycin have been compared for their in-vitro interaction with bactericidal killing by human neutrophils. The mechanism of this interaction was studied in two ways. First, the target organisms (Staphylococcus aureus and Pseudomonas aeruginosa) were incubated for 60 min with josamycin, erythromycin or control buffer prior to use in a human polymorphonuclear neutrophil (PMN) killing assay. Second the macrolides were added directly to acellular killing systems mimicking those acting inside the phagolysosome; oxygen-independent systems were obtained from a crude granule extract of PMN and oxygen-dependent systems consisted either of a mixture of xanthine plus xanthine oxidase or of a solution of H2O2. Whereas josamycin-pretreated P. aeruginosa were twice as sensitive to killing by PMN than were control cells, this was not the case for S. aureus. Both oxidant generating systems were more effective in destroying S. aureus in the presence of josamycin (3 and 30 mg/l). Erythromycin showed a similar synergy but only with the xanthine plus xanthine oxidase system. This synergy was observed with neither of the O2-independent systems for S. aureus, nor with any acellular system for P. aeruginosa. These data suggest that at least two kinds of mechanism may explain the bactericidal synergy observed between macrolides and PMN. The first (for macrolide-resistant species such as P. aeruginosa) could be due to alterations in the bacteria by the antibiotics, while the second (for macrolide-sensitive species such as S. aureus) could be based upon an as yet unexplained transformation of the molecules by reactive oxygen species into more "toxic" forms. These differences between josamycin and erythromycin could arise from differences in their chemical structure.

Clinical efficacy and tolerance of two new macrolides, clarithromycin and josamycin, in the treatment of patients with acute exacerbations of chronic bronchitis.

The efficacy and safety of oral 500 mg clarithromycin given twice daily and 500 mg josamycin given three times daily were compared for up to 14 days in the treatment of 103 out-patients with acute exacerbations of chronic bronchitis in an open, randomized study. The predominant pathogens isolated were Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus. Clinical cures were experienced by 85% of the clarithromycin-treated patients, with an additional 10% showing improvement. In josamycin-treated patients, 76% of the patients experienced clinical cures and an additional 19% showed improvement. Eradication of the causative pathogen occurred in approximately 95% of the patients in both treatment groups. Adverse events, which all involved the gastro-intestinal tract and were mild, transient and probably related to therapy, were experienced by 5.8% and 7.8%, respectively, of the clarithromycin- and josamycin-treated patients. It is concluded that clarithromycin given twice daily is as effective as josamycin given three times daily.